Medicinal use of serrulatane diterpenes

ABSTRACT

The present disclosure is drawn to methods of treating diseases or disorders, and uses of preparing medicaments. The methods of administering and/or preparation of medicaments can include the use of a compound of Formula (I) disclosed herein, or a geometric isomer, stereoisomer, and/or pharmaceutically acceptable salt or solvate thereof.

The present application is a Continuation in Part of U.S. application Ser. No. 16/330,676 filed on Mar. 5, 2019, which is a National Stage of International Application No. PCT/AU2017/050972 filed on Sep. 7, 2017, which claims priority to foreign application No. AU2016903585 filed on Sep. 7, 2016, each of which are incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to terpenes and uses thereof. In particular, the invention relates to serrulatane diterpenes and to the use of such compounds in the treatment of diseases and disorders such as cancer.

Throughout this application, various publications are referred to by first author and year of publication. Full citations for these publications, in the order they appear in the application, are presented in a References section immediately before the claims.

BACKGROUND OF THE INVENTION

Plant secondary metabolites have been a major source of medicinal substances providing lead compounds for drug discovery and development.

Plants produce resins and exudates containing secondary metabolites to protect young flower and leaf buds against bacteria and fungi, and damage from sunlight. Accordingly, compounds from plant resins and exudates frequently possess antimicrobial and antioxidant/free radical scavenging activities. Worker honey bees (Apis mellifera) collect plant resins/exudates of young shoots and buds from certain trees and shrubs. The resulting mixture is called propolis, or so called bee glue. Propolis is a complex resinous substance and a rich source of bioactive substances. It is used by the bees to mix with wax to seal cracks and holes in their hives and as a disinfectant to protect against microbial infections. The medicinal properties of propolis have been exploited by man since Ancient civilizations.

Currently, propolis is extensively available as a natural health product, and is widely used in cosmetics. However, its modern use in medicine is limited, largely due to the wide variations in chemical compositions arising from honey bees collecting from different or a mixture of plant sources. The composition of propolis is dependent upon the surrounding flora to which the bees have access, and as such, differences in flora may result in differences in propolis compositions. For example, it is known that flavonoids are the major pharmacologically active compounds in European propolis, polyprenylated benzophenones are the main substances in Cuban and Venuzuelan propolis, and prenylated cinnamic acid derivatives are predominant in Brazilian propolis.

Recognition of the botanical origin of the propolis produced by honey bees enables beehives to be placed in favourable locations such that propolis from a single botanical source may be produced to enable manufacture of medicines of high quality and efficacy.

The medicinal uniqueness of propolis is determined by the selective collecting ability of honey bees, as they can recognise natural materials that are relatively non-polar and have antibiotic properties. As reported, the common source of propolis is leaf and flower bud exudates, which are of high antibiotic character in order to protect the delicate growing of plant tissue from attack by microorganisms. It has also been reported that honey bees collect exudates from wounded or diseased plant tissues. Such sources are potentially rich in antibiotic substances produced by plants in response to wounding or attack from insects, microorganisms and viruses.

Medicinal properties of a propolis are associated with the biological activities of the individual plant resins/exudates. It is not clear from previous studies whether the bees simply collect a plant material that is known as propolis, or if there is metabolic modification or addition from the bees. However, there does not appear to be evidence of significant amounts of material added from honey bees, or strong evidence for metabolic transformation. Thus, a better understanding is required regarding the composition of propolis in specific geographical locations to be able to utilize it to its full benefit in the development of new agents in the treatment of diseases and disorders such as cancer.

SUMMARY OF THE INVENTION

In work leading up to the present invention a survey of propolis samples isolated from Kangaroo Island (South Australia) was conducted. Surprisingly, the Inventors found that unlike other propolis which commonly contains flavonoids as active constituents, Kangaroo Island propolis from the Myoporum genus of plants, in particular Myoporum insulare, contains serrulatane diterpenes. Thus, the present invention relates to compounds isolated from the resins/exudates of leaves and leaf buds of Myoporum insulare, propolis sourced from the same plant and uses thereof.

Myoporum insulare species is a shrub occurring on ridges and coastal cliffs in Australia. Common names include boobialla, native juniper and, in Western Australia, blueberry tree. Myoporum is a genus of approximately 30 species, of which sixteen are found in Australia. Myoporum insulare species has a variable growth habitat and may be a dense or an open shrub or a small tree up to 6 metres. The leaves are lance-shaped to elliptical, 30-100 mm long by 10-20 mm wide with glossy green colour. The flowers occur in groups of up to 8 in the leaf axils in late spring and summer and are usually white or occasionally pale pink.

The Inventors have isolated five major compounds, namely compounds 1 to 5, from propolis obtained from Myoporum insulare. Compounds 1 to 5 are shown in Table 1. The conventional numbering for serrulatane compounds is shown with respect to Compound 1. In general, serrulatane diterpenes have the (1R,4S)-configuration (Ghisalberti, 1994).

Compound 1 gives a derivative, Compound 6, on oxidation. Further in regard to derivatization, the compounds can also be acylated, alkylated, alkenylated or benzoylated through the free hydroxyl groups. For example, compounds 1 and 2 can be acetylated to give derivatives 7 and 8 while benzoylation can give derivatives 9 and 10.

TABLE 1 Representative serrulatane diterpenoid compounds.

1

2

3

4

5

TABLE 2 Serrulatane diterpenoid derivatives.

 6

 7

 8

 9

10

11

12

Representative compounds were evaluated for pharmacological activity and were found to be useful in modulating a number of diseases or disorders. For example, the compounds were found to inhibit cancer cell proliferation and showed moderate to strong inhibition of various targets associated with cancer pathology. This indicates that the serrulatane diterpene compounds of the present disclosure may be useful as therapeutic agents e.g. for the treatment of cancer. The compounds were also found to effectively inhibit inflammatory activity and modulate immune response, showing moderate to strong inhibition of COX1 and COX2, the release of proinflammatory cytokines, lipoxygenase and the nuclear transcription factor NF-κB. Therefore, the serrulatane diterpenes described herein provide a potentially attractive lead for pharmaceutical research and development and as biological tools for further understanding the pathophysiology of diseases and disorders such as cancer, and diseases and disorders associated with inflammation, such as skin inflammatory conditions.

Accordingly, in a first aspect, there is provided a method of treating a cancer, the method comprising administering a therapeutically effective amount of a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition including said compounds, to a subject in need thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl. OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH.

According to a second aspect, there is provided use of a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

in the preparation of a medicament for treating a cancer.

According to a third aspect, there is provided use of a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

in treating a cancer.

According to a fourth aspect, there is provided a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

for use in treating a cancer.

According to a fifth aspect, there is provided a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH.

According to a sixth aspect, there is provided a pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to the fifth aspect and a pharmaceutically acceptable excipient.

According to a seventh aspect, there is provided a pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to the fifth aspect, or a mixture thereof, and a pharmaceutically acceptable excipient.

According to an eighth aspect, there is provided a compound as defined in the fifth aspect isolated from propolis, wherein the propolis originates from plants of the Myoporum genus.

According to a ninth aspect, there is provided a compound as defined in the fifth aspect isolated from the resin, gum or exudate of Myoporum genus.

According to a tenth aspect, there is provided a method of treating a disease or disorder, the method comprising administering a therapeutically effective amount of a compound according to the fifth aspect or a composition according to the sixth or seventh aspect.

According to an eleventh aspect, there is provided use of a compound according to the fifth aspect in the preparation of a medicament for treating a disease or disorder.

According to a twelfth aspect, there is provided use of a compound according to the fifth aspect or a composition according to the sixth aspect or seventh aspect in treating a disease or disorder.

According to a thirteenth aspect of the present invention, there is provided a compound according to the fifth aspect or a composition according to the sixth aspect or seventh aspect for use in treating a disease or disorder.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows representative ¹H NMR (400 MHz, CDCl₃) spectrum of the 4:1 mixture of mono-methylated products (compounds 11 and 12).

FIG. 2 shows representative ¹³C NMR (100 MHz, CDCl₃) spectrum of the 4:1 mixture of mono-methylated products (compounds 11 and 12).

FIG. 3 to FIG. 6 show example binding curves for Compound 1 against epigenetic targets.

FIG. 3 shows ASH1L (h) bromodomain binding curve.

FIG. 4 shows CECR2(h) bromodomain binding curve.

FIG. 5 shows SP140 (h) bromodomain binding curve.

FIG. 6 shows UHRF1(108-286) (h) binding curve.

FIG. 7 shows binding against 5-lipoygenase for Compound 1 (circles, IC₅₀=2.3 μM) and a reference compound NSA (squares, IC₅₀=0.64 μM).

FIG. 8 shows binding against lipid peroxidase for Compound 1 (circles, IC₅₀=1.47 μM) and a reference compound N-propyl gallate (squares, IC₅₀=206 μM).

FIG. 9 shows binding against monoamine oxidase (MAO-A) for Compound 1 (circles, IC₅₀=6.47 μM) and a reference compound clorgyline (squares, IC₅₀=1.43 nM).

FIG. 10 shows transcription response and binding against NF-kB for Compound 1 (circles, IC₅₀=1.36 μM) and a reference compound cyclosporin (squares, IC₅₀=0.029 μM).

FIG. 11 shows proliferation response when cell line MDA-MB-415 is treated with Compound 1.

FIG. 12 shows proliferation response when cell line RKO-AS45-1 is treated with Compound 1.

FIG. 13 shows proliferation response when cell line SW480 is treated with Compound 1.

FIG. 14 shows proliferation response when cell line 639-V is treated with Compound 1.

FIG. 15 shows proliferation response when cell line Hs 729 is treated with Compound 1.

FIG. 16 shows proliferation response when cell line Hs 852.T is treated with Compound 1.

FIG. 17 shows proliferation response when cell line HCT-8 is treated with Compound 1.

FIG. 18 shows proliferation response when cell line IM-9 is treated with Compound 1.

FIG. 19 shows the response when the cell line HREC is treated with Compound 1 in an OncoPanel Normal Cell Proliferation Assay.

FIGS. 20A to 20D show various dose-responsive effects of an example compound on A) CCL2 release in human keratinocytes, B) IL-8 release in human keratinocytes, C) IL-17A release in human PBMC inflammation, and D) IL-2 and IL-4 release in T-cell inflammation, respectively.

Specific embodiments of the disclosure are described below. It will be appreciated that these embodiments are illustrative and not restrictive.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a method of treating a cancer, the method comprising administering a therapeutically effective amount of a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition including said compounds, to a subject in need thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl. OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

Preferably, the compound is a compound of Formula (Ib):

Preferably, the compound is a compound of Formula (Ic):

Preferably, the compound is a compound of Formula (Id):

Preferably, the compound is a compound of Formula (Ie):

Preferably, the compound is a compound of Formula (If):

Preferably, the compound is a compound of Formula (Ig):

Preferably, the compound is a compound of Formula (Ih):

Preferably, the compound is a compound of Formula (Ii):

Preferably, A-B is CH═C.

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

Preferably, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

The cancer of the central nervous system is preferably a glioma. Preferably, the cancer of the central nervous system is a medulloblastoma. The cancer of the central nervous system is preferably a neuroblastoma.

Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

While it is possible that, for use in therapy a therapeutically effective amount of the compounds as defined herein, or a pharmaceutically acceptable salt or solvate thereof, may be administered as the raw chemical; in the first aspect of the present invention the active ingredient is administered as a pharmaceutical composition. Thus, the present invention also contemplates a pharmaceutical composition comprising a compound of formula (I) to (Ii), or a pharmaceutically acceptable salt or a solvate thereof, and a pharmaceutically acceptable excipient. The excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

When applicable, the compounds of the present invention, including the compounds of formula (I) to (Ii) may be in the form of and/or may be administered as a pharmaceutically acceptable salt.

As used herein the term “pharmaceutically acceptable salt” refers to salts which are toxicologically safe for systemic administration. The pharmaceutically acceptable salts may be selected from alkali or alkaline earth metal salts, including, sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethyl ammonium, methylamine, dimethylamine, trimethylamine, tri ethyl amine, ethylamine, triethanolamine and the like.

As used herein the term “pharmaceutically acceptable excipient” refers to a solid or liquid filler, carrier, diluent or encapsulating substance that may be safely used in administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers or excipients may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

As used herein, the term “solvate” refers to a complex of variable stoichiometry formed by a solute (in this disclosure, a compound of formula (I) to (Ii) or a salt or physiologically functional derivative thereof) and a solvent. Such solvents for the purpose of the disclosure may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol and acetic acid. In particular the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include, without limitation, water, ethanol, acetic acid, glycerol, liquid polyethylene glycols and mixtures thereof. A particular solvent is water.

Administration of compounds of the formula (I) to (Ii) may be in the form of a “prodrug”. A prodrug is an inactive form of a compound which is transformed in vivo to the active form. Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question.

The compounds of the present disclosure may be suitable for the treatment of diseases in a human or animal subject. In one example, the subject is a mammal including a human, horse, dog, cat, sheep, cow, or primate. In another example, the subject is a human. In a further example, the subject is not a human. The terms “subject” and “patient” are used interchangeably herein.

As used herein, the term “effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician.

Furthermore, the term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. The term also includes within its scope amounts effective to prevent a disease, disorder, or side effect.

As used herein the term “treatment” refers to defending against or inhibiting a symptom, treating a symptom, delaying the appearance of a symptom, reducing the severity of the development of a symptom, and/or reducing the number or type of symptoms suffered by an individual, as compared to not administering a pharmaceutical composition comprising a compound of the invention. The term also includes within its scope prevention of a disease, disorder, or side effect. The term “treatment” encompasses use in a palliative setting

The antitumor effect of the compounds of the present disclosure may be applied as a sole therapy or as a combination therapy i.e. where two or more serrulatane diterpenoids may be administered in combination. Therapy may also involve administration of a mixture of two or more serrulatane diterpenoids. Therapy may involve, in addition, administration of one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment. In the field of medical oncology it is normal practice to use a combination of different forms of treatment to treat each patient with cancer such as a combination of surgery, radiotherapy and/or chemotherapy. In particular, it is known that irradiation or treatment with antiangiogenic and/or vascular permeability reducing agents can enhance the amount of hypoxic tissue within a tumour. Therefore the effectiveness of the compounds of the present invention may be improved by conjoint treatment with radiotherapy and/or with an antiangiogenic agent.

The individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present disclosure is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly. It will be understood that the scope of combinations of the compounds of this invention with other anti-neoplastic agents includes in principle any combination with any pharmaceutical composition useful for treating cancer.

When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation and may be formulated for administration. When formulated separately they may be provided in any convenient formulation, conveniently in such a manner as are known for such compounds in the art.

Pharmaceutical compositions of the present disclosure may be formulated for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Therefore, the pharmaceutical compositions of the invention may be formulated, for example, as tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions. Such pharmaceutical formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Such pharmaceutical formulations may be prepared as enterically coated granules, tablets or capsules suitable for oral administration and delayed release formulations.

When a compound is used in combination with a second therapeutic agent active against the same disease, the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

Also disclosed herein is use of a compound of Formula (I),

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl. OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

in the preparation of a medicament for treating a cancer.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

Preferably, the compound is a compound of Formula (Ib):

Preferably, the compound is a compound of Formula (Ic):

Preferably, the compound is a compound of Formula (Id):

Preferably, the compound is a compound of Formula (Ie):

Preferably, the compound is a compound of Formula (If):

Preferably, the compound is a compound of Formula (Ig):

Preferably, the compound is a compound of Formula (Ih):

Preferably, the compound is a compound of Formula (Ii):

Preferably, A-B is CH═C.

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

Preferably, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

The cancer of the central nervous system is preferably a glioma. Preferably, the cancer of the central nervous system is a medulloblastoma. The cancer of the central nervous system is preferably a neuroblastoma.

Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

Also disclosed herein is use of a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt, or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl. OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

in treating a cancer.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

Preferably, the compound is a compound of Formula (Ib):

Preferably, the compound is a compound of Formula (Ic):

Preferably, the compound is a compound of Formula (Id):

Preferably, the compound is a compound of Formula (Ie):

Preferably, the compound is a compound of Formula (If):

Preferably, the compound is a compound of Formula (Ig):

Preferably, the compound is a compound of Formula (Ih):

Preferably, the compound is a compound of Formula (Ii):

Preferably, A-B is CH═C.

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

Preferably, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

The cancer of the central nervous system is preferably a glioma. Preferably, the cancer of the central nervous system is a medulloblastoma. The cancer of the central nervous system is preferably a neuroblastoma.

Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

Also disclosed herein is a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

for use in treating a cancer.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

Preferably, the compound is a compound of Formula (Ib):

Preferably, the compound is a compound of Formula (Ic):

Preferably, the compound is a compound of Formula (Id):

Preferably, the compound is a compound of Formula (Ie):

Preferably, the compound is a compound of Formula (If):

Preferably, the compound is a compound of Formula (Ig):

Preferably, the compound is a compound of Formula (Ih):

Preferably, the compound is a compound of Formula (Ii):

Preferably, A-B is CH═C.

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

Preferably, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

The cancer of the central nervous system is preferably a glioma. Preferably, the cancer of the central nervous system is a medulloblastoma. The cancer of the central nervous system is preferably a neuroblastoma.

Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

Also disclosed herein is a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl. OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

Preferably, the compound is a compound of Formula (Ib):

Preferably, the compound is a compound of Formula (Ic):

Preferably, the compound is a compound of Formula (Id):

Preferably, the compound is a compound of Formula (Ie):

Preferably, the compound is a compound of Formula (If):

Preferably, the compound is a compound of Formula (Ig):

Preferably, the compound is a compound of Formula (Ih):

Preferably, the compound is a compound of Formula (Ii):

Preferably, A-B is CH═CH.

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably selected from the group consisting of:

Preferably, the compound is selected from the group consisting of:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

The compound is preferably:

Preferably, the compound is:

Also disclosed herein is a pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, as described herein, and a pharmaceutically acceptable excipient.

Also disclosed herein is a pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, as described herein, or a mixture thereof, and a pharmaceutically acceptable excipient.

The compounds or pharmaceutical compositions disclosed above are preferably for use as a medicament.

The compounds or pharmaceutical compositions disclosed above are preferably for use in therapy.

Also disclosed is a compound described above isolated from propolis, wherein the propolis originates from plants of the Myoporum genus. Preferably, the propolis originates from Myoporum insulare.

Also disclosed is a compound described above isolated from the resin, gum or exudate of Myoporum genus. Preferably, the compound is isolated from the resin, gum or exudate of Myoporum insulare.

Also disclosed is a method of treating a disease or disorder, the method comprising administering a therapeutically effective amount of a compound or a composition described herein.

Also disclosed is use of a compound described herein in the preparation of a medicament for treating a disease or disorder.

Also disclosed herein is use of a compound or a composition described herein in treating a disease or disorder.

Also disclosed herein is a compound or a composition described herein for use in treating a disease or disorder.

Preferably, the disease or disorder is selected from the group consisting of: acute coronary syndrome, an aging-related disease or disorder; an allergic disease or a related condition; Alzheimer's disease, atherosclerosis, an autoimmune disease; a bacterial infection, cancer; dementia, depression or a related condition; diabetes; dyslipidemia, hyperlipidemia, hypertension, itchytosis, an immune disease; a metabolic disease or disorder; a neurological disease or disorder; obesity; Parkinson's disease; pain; rheumatoid arthritis, a disease or disorder associated with inflammation, such as skin inflammatory conditions, and a skin disease or disorder.

The disease or disorder is preferably cancer.

Preferably, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

The cancer of the central nervous system is preferably a glioma. Preferably, the cancer of the central nervous system is a medulloblastoma. The cancer of the central nervous system is preferably a neuroblastoma.

Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma.

Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

The skin disease or disorder is preferably selected from the group consisting of: eczema, psoriasis, acne, a wound, a scar, inflammation including psoriasis and atopic dermatitis, a burn, sunburn, skin damage and skin irritation.

Representative compounds were evaluated for pharmacological activity and were found to be useful in modulating a number of diseases or disorders. For example, the compounds were found to inhibit cancer cell proliferation and showed moderate to strong inhibition of various targets associated with cancer pathology. The compounds were also found to effectively inhibit inflammatory activity and modulate immune response, showing moderate to strong inhibition of COX1 and COX2, the release of proinflammatory cytokines, lipoxygenase and the nuclear transcription factor NF-κB. Further discussion of compound activity is presented below in the Examples.

Example Embodiments

1. A method of treating a cancer, the method comprising administering a therapeutically effective amount of a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition including said compounds, to a subject in need thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl. OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH.

2. Use of a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl. OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

in the preparation of a medicament for treating a cancer.

3. Use of a compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl. OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

in treating a cancer.

4. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl; and

A-B is CH═C or CH₂—CH.

5. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph; and

A-B is CH═C or CH₂—CH.

6. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

7. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CH₂;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

8. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CHOH;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

9. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

10. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ia):

11. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ib):

12. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ic):

13. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Id):

14. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ie):

15. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (If):

16. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ig):

17. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ih):

18. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ii):

19. The method or use according to any one of example embodiments 1 to 16, wherein A-B is CH═C.

20. The method according to any one of example embodiments 1 to 19 or the use according to any one of example embodiments 2 to 19, wherein the compound is selected from the group consisting of:

21. The method or use according to example embodiment 20, wherein the compound is selected from the group consisting of:

22. The method or use according to example embodiment 21, wherein the compound is selected from the group consisting of:

23. The method or use according to example embodiment 22, wherein the compound is selected from the group consisting of:

24. The method or use according to example embodiment 23, wherein the compound is:

25. The method or use according to example embodiment 23, wherein the compound is:

26. The method or use according to example embodiment 23, wherein the compound is:

27. The method or use according to example embodiment 23, wherein the compound is:

28. The method or use according to example embodiment 23, wherein the compound is:

29. The method or use according to example embodiment 23, wherein the compound is:

30. The method according to any one of example embodiments 1 to 29 or the use according to any one of example embodiments 2 to 29, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

31. The method or use according to example embodiment 30, wherein the cancer of the central nervous system is a glioma.

32. The method or use according to example embodiment 30, wherein the cancer of the central nervous system is a medulloblastoma.

33. The method or use according to example embodiment 30, wherein the cancer of the central nervous system is a neuroblastoma.

34. The method or use according to example embodiment 30, wherein the lung cancer is a non-small cell lung cancer.

35. The method or use according to example embodiment 30, wherein the lung cancer is a small cell lung cancer.

36. The method or use according to example embodiment 30, wherein the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

37. The method or use according to example embodiment 30, wherein the sarcoma is a liposarcoma, rhabdomyosarcoma, or fibrosarcoma.

38. The method or use according to example embodiment 30, wherein the sarcoma is a soft tissue sarcoma.

39. The method or use according to example embodiment 38, wherein the soft tissue sarcoma is a soft tissue osteosarcoma.

40. The method according to example embodiment 30, wherein the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

41. A compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH.

42. A compound of Formula (I),

a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; and

A-B is CH═C or CH₂—CH,

for use in treating a cancer.

43. The compound according to example embodiment 41 or example embodiment 42,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC(O)C₁₋₅alkyl, OC(O)Ph or OC(O)C₂₋₅alkenyl; and

A-B is CH═C or CH₂—CH.

44. The compound according to example embodiment 41 or example embodiment 42,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph; and

A-B is CH═C or CH₂—CH.

45. The compound according to example embodiment 41 or example embodiment 42,

wherein:

X is CH₂, CHOH or C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

where X is CH₂, CHOH or C(O), R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

46. The compound according to example embodiment 41 or example embodiment 42,

wherein:

X is CH₂;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

47. The compound according to example embodiment 41 or example embodiment 42,

wherein:

X is CHOH;

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

48. The compound according to example embodiment 41 or example embodiment 42, wherein:

X is C(O);

R₁ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₂ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O;

and no more than one of R₁, R₂ and R₃ can be H;

R₄ is H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph;

or X and R₃ form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄ is respectively OH or OC₁₋₅alkyl or OC(O)C₁₋₅alkyl; and

A-B is CH═C or CH₂—CH.

49. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ia):

50. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ib):

51. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ic):

52. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Id):

53. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ie):

54. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (If):

55. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ig):

56. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ih):

57. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ii):

58. The compound according to any one of example embodiments 41 to 57, wherein A-B is CH═C.

59. The compound according to any one of example embodiments 41 to 58, wherein the compound is selected from the group consisting of:

60. The compound according to example embodiment 59, wherein the compound is selected from the group consisting of:

61. The compound according to example embodiment 60, wherein the compound is selected from the group consisting of:

62. The compound according to example embodiment 61, wherein the compound is selected from the group consisting of:

63. The compound according to example embodiment 62, wherein the compound is:

64. The compound according to example embodiment 62, wherein the compound is:

65. The compound according to example embodiment 62, wherein the compound is:

66. The compound according to example embodiment 62, wherein the compound is:

67. The compound according to example embodiment 62, wherein the compound is:

68. The compound according to example embodiment 62, wherein the compound is:

69. The compound according to any one of example embodiments 42 to 68, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

70. The compound according to example embodiment 69, wherein the cancer of the central nervous system is a glioma.

71. The compound according to example embodiment 69, wherein the cancer of the central nervous system is a medulloblastoma.

72. The compound or use according to example embodiment 69, wherein the cancer of the central nervous system is a neuroblastoma.

73. The compound according to example embodiment 69, wherein the lung cancer is a non-small cell lung cancer.

74. The compound according to example embodiment 69, wherein the lung cancer is a small cell lung cancer.

75. The compound according to example embodiment 69, wherein the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

76. The compound according to example embodiment 69, wherein the sarcoma is a liposarcoma, rhabdomyosarcoma, or fibrosarcoma.

77. The compound according to example embodiment 69, wherein the sarcoma is a soft tissue sarcoma.

78. The compound according to example embodiment 77, wherein the soft tissue sarcoma is a soft tissue osteosarcoma.

79. The compound according to example embodiment 69, wherein the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

80. A pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to example embodiment 41 or any one of example embodiments 43 to 68, and a pharmaceutically acceptable excipient.

81. A pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to example embodiment 41 or any one of example embodiments 43 to 68, or a mixture thereof, and a pharmaceutically acceptable excipient.

82. The compound according to example embodiment 41 or any one of example embodiments 43 to 68, or the pharmaceutical composition according to example embodiment 80 or example embodiment 81, for use as a medicament.

83. The compound according to example embodiment 41 or any one of example embodiments 43 to 68, or the pharmaceutical composition according to example embodiment 80 or example embodiment 81, for use in therapy.

84. A compound as defined in any one of example embodiments 42 to 68 isolated from propolis, wherein the propolis originates from plants of the Myoporum genus.

85. The compound according to example embodiment 84, wherein the propolis originates from Myoporum insulare.

86. A compound as defined in example embodiment 41 or any one of example embodiments 43 to 68 isolated from the resin, gum or exudate of Myoporum genus.

87. The compound according to example embodiment 86 isolated from the resin, gum or exudate of Myoporum insulare.

88. A method of treating a disease or disorder, the method comprising administering a therapeutically effective amount of a compound example embodiment 41 or any one of example embodiments 43 to 68 or a composition according to example embodiment 80 or example embodiment 81.

89. Use of a compound according to example embodiment 41 or any one of example embodiments 43 to 68 in the preparation of a medicament for treating a disease or disorder.

90. Use of a compound according to example embodiment 41 or any one of example embodiments 43 to 68 or a composition according to example embodiment 80 or example embodiment 81 in treating a disease or disorder.

91. A compound according to example embodiment 41 or any one of example embodiments 43 to 68 or a composition according to example embodiment 80 or example embodiment 81 for use in treating a disease or disorder.

92. The method according to example embodiment 88 or use according to example embodiment 89 or example embodiment 90 or compound according to example embodiment 91, wherein the disease or disorder is selected from the group consisting of: acute coronary syndrome, an aging-related disease or disorder; an allergic disease or a related condition; Alzheimer's disease, atherosclerosis, an autoimmune disease; a bacterial infection, cancer; dementia, depression or a related condition; diabetes; dyslipidemia, hyperlipidemia, hypertension, itchytosis, an immune disease; a metabolic disease or disorder; a neurological disease or disorder; obesity; Parkinson's disease; pain; rheumatoid arthritis, a disease or disorder associated with inflammation, such as skin inflammatory conditions, and a skin disease or disorder.

93. The method or use or compound according to example embodiment 92, wherein the disease or disorder is cancer.

94. The method or use or compound according to example embodiment 93, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

95. The method or use or compound according to example embodiment 94, wherein the cancer of the central nervous system is a glioma.

96. The method or use or compound according to example embodiment 94, wherein the cancer of the central nervous system is a medulloblastoma.

97. The method or use or compound according to example embodiment 94, wherein the cancer of the central nervous system is a neuroblastoma.

98. The method or use or compound according to example embodiment 94, wherein the lung cancer is a non-small cell lung cancer.

99. The method or use or compound according to example embodiment 94, wherein the lung cancer is a small cell lung cancer.

100. The method or use or compound according to example embodiment 94, wherein the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

101. The method or use or compound according to example embodiment 94, wherein the sarcoma is a liposarcoma, rhabdomyosarcoma, or fibrosarcoma.

102. The method or use or compound according to example embodiment 94, wherein the sarcoma is a soft tissue sarcoma.

103. The method or use or compound according to example embodiment 102, wherein the soft tissue sarcoma is a soft tissue osteosarcoma.

104. The method or use or compound according to example embodiment 94, wherein the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

105. The method or use or compound according to example embodiment 92, wherein the skin disease or disorder is selected from the group consisting of: eczema, psoriasis, acne, a wound, a scar, inflammation including psoriasis and atopic dermatitis, a burn, sunburn, skin damage and skin irritation.

EXAMPLES

In order to better understand the nature of the invention a number of examples will now be described as follows:

Experimental

Materials

Honey bees observed collecting from the boobialla trees and shrubs (Myoporum insulare) were captured in plastic tubes, capped and frozen. Sections of the bee hind legs holding propolis were cut and pooled. Plant samples were obtained and dried at 40° C. in a ventilated oven (Thermoline, NSW Australia) a few days to provide voucher specimens for identification by botanist, A/Prof Murray Henwood, John Ray Herbarium, University of Sydney, Sydney, Australia, and registered as Myoporum insulare voucher number Duke-131202-01. Resin extracted from plant samples was stored at −20° C. until analysis or further processing.

Propolis was obtained from apiary sites with from 1 to 10 colonies of European honey bee (Apis mellifera ligustica) in standard 10-frame box hives, each fixed with a propolis mat under the hive cover lid. The propolis samples were collected and provided by a few beekeepers in eight apiary sites located in different parts of Kangaroo Island, South Australia. These sites were located in the vicinity of Hanson Bay, Vivonne Bay, Flour Cask Bay, Island Beach, Kingscote, Brownlow, Rainy Creek and Eleanor River. These locations are known to be a habitat for the plant Myoporum insulare on Kangaroo Island. This provided 23 propolis samples which, based on TLC analysis and ¹H NMR techniques, were estimated to be at least 90% sourced from M. insulare. The samples were stored at −20° C. until further analysis and processing.

Thin layer chromatography sheets precoated with silica gel 60 F₂₅₄ and silica gel 60H for normal-phase short-column vacuum chromatography (NPSCVC) were purchased from Merck. TLC plates were visualized with a UVGL-58 mineral-light lamp, Multiband UV-2544/366.

All the chemicals used in the isolation and synthesis, including deuterated NMR solvents such as d-chloroform, d₄-methanol and d₆-dimethyl sulfoxide were purchased from Novachem Pty Ltd (Collingwood, Vic, Australia). Solvents including hexane, dichloromethane, ethyl acetate, isopropanol, ethanol, methanol, and acetic acid were of analytical grade and purchased from Chem Supply, Gillman, SA, Australia.

General Methods

Rotavapor model R-114 rotary evaporator with a water bath temperature ranging between 40-60° C. was used to evaporate the solvent fraction. Vacuum pump V-700 or Vacuubrand MD 4C NT diaphragm pump (Vacuubrand GMBH, Wertheim, Germany) with vacuum controller V-800 or V-850 is used. Final drying is carried out by a Napco 5831 vacuum oven (NAPCO, Salt Lake City, USA) using a DirectTorr vacuum pump (Sargent-Welch, Buffalo, USA).

Isocratic analytical HPLC was performed on a Shimadzu UFLC, LC-20AD pump, SIL-20A HT autosampler, with a Hewlett-Packard Column, NUCLEOSIL 100 C₁₈, 5 μm, 4 mm×125 mm, injection volume 20 μl, eluted at 1 ml/min and detected at 230 nm with a UV-Vis detector (Shimadzu SPD-20A). The column was eluted with methanol-water-acetic acid (65:34.8:0.2).

Gradient analytical HPLC was performed on a Shimadzu Nexera X2 LC-30AD system, SIL-30 autosampler, with a Hewlett-Packard Column, NUCLEOSIL 100 C₁₈, 5 μm, 4 mm×125 mm, injection volume 10 μl, eluted at 1 ml/min and detected at 320 nm with a SPD-M30A UV-Vis diode array detector. The column was eluted with a gradient system made up of methanol (phase A), and methanol:water:acetic acid (40:59.8:0.2) (phase B). A solvent gradient was applied as follows: 0-2 min: 32-50% A, 2-9 min: 50-90% A, 9-12 min: 90-100% A, 12-14 min, 100% A, and finally 14-15 min: 100-32% A, maintained over 5 min.

¹H and ¹³C Nuclear magnetic resonance (NMR) analyses were carried out on Varian 400 MHz System with a SMS autosampler (Palo Alto, Calif., USA). Chemical shifts (5) of peaks in NMR spectra are reported in parts per million (ppm) and are referenced to tetramethylsilane (Si(CH₃)₄, TMS). ¹H NMR data is reported as chemical shift (5), relative integral, multiplicity (abbreviations: s=singlet, d=doublet, I=triplet, id=triplet of doublets, q=quartet, dd=doublet of doublets, ddd=doublet of doublet of doublets, bs=broad singlet, hi=broad triplet), coupling constant (Jin Hz) and assignment. ¹³C NMR data is reported as chemical shift (5) and assignment.

For low resolution ESI-MS, the samples were analyzed by Finnigan Polaris Ion Trap MS/MS system (Finnigan Corporation, San Jose, USA) using an Xcalibur 1.2 data system, in Electrospray Ionization (ESI) negative and positive modes using infusion technique.

High resolution ESI-MS were measured on a Bruker Daltonics Apex Ultra Fourier Transform Ion Cyclotron Resonance 7 Tesla Mass Spectrometer, mass spectrometry unit, School of Chemistry, The University of Sydney.

Determination of the ¹H-NMR Chemical Profiles of the Plant and Propolis Specimens

Young leaf shoots (25 g), bee hind leg (0.01 g) and beehive propolis (1.0 g) were extracted with dichloromethane at room temperature for 15 min. The extracts were filtered, dried under reduced pressure and analyzed by ¹H-NMR and HPLC. Samples were found to contain serrulatane diterpenes. Propolis and plant samples were subsequently selected for isolation of the components.

Preparation of Myoporum insulare Resin Extract

Fresh young leaves and young shoot tips were harvested from several Myoporum insulare trees. The leaves were stored in plastic bags on ice until processing within 5 days. To extract the external resin/exudate the leaves and shoot tips (4 Kg) were mixed with dichloromethane (4 litres) and after 1 hour standing the dichloromethane extract was filtered and the dichloromethane was removed from the filtrate by evaporation under reduced pressure using a rotary evaporator to give a brown partially solid dried resin (12.5 g, yield 0.31%).

Isolation and Identification of Serrulatane Diterpenes from Myoporum insulare Propolis from Kangaroo Island.

General Method

Propolis (100 g) was extracted with dichloromethane at room temperature with stirring for 1 hr. The extract was subjected to purification using normal-phase short column vacuum chromatography (NPSCVC). A step-wise gradient of mobile phase (2×100 mL) consisting of dichloromethane (DCM) and ethyl acetate (EtOAc) at 0, 1, 2, 4, 8, 10, 15, 20, 50 and 100% was employed to elute the components which were analysed by TLC and NMR. Further purification of the compounds, if required, was subsequently carried out on the same NPSCVC with different mobile phases consisting of either hexane and EtOAc or hexane and isopropanol. Structures and identity of these purified compounds were characterized by ¹H- and ¹³C-NMR and mass spectrometry including high resolution mass spectrometry. Detailed structural analyses of the isolated compound were also carried out when needed by 2D-NMR using Gradient Heteronuclear Multiple Bond Coherence (GHMBC). The major serrulatane diterpenoid was determined to be 7,8,18-trihydroxyserrulat-14-ene (1).

Isolated Serrulatane Diterpenes from the Plant, Myoporum insulare.

7,8,18-trihydroxyserrulat-14-ene (1)

Yellowish liquid, yield 50 mg, [α]_(D) ²⁰ −27.3° (C=1.0, CHCl₃). UV (CH₃OH) λ_(max) nm (log ε) 279 (3.37) and 320 (2.49). IR (v): 3381, 2924, 2864, 1625, 1581, 1448 cm⁻¹. HRESIMS: 341.2089 m/z [M+Na]⁺ (calcd 341.2087) C₂₀H₃₀O₃. ¹H NMR (400 MHz, CDCl₃) δ: 6.56, (1H, 5, H-5), 5.04 (1H, bt, J=7.0 Hz, H-14), 3.63 (2H, d, J=6.2 Hz, H-18), 3.07 (1H, pentet of d, J=6.8, 1.6 Hz, H-1), δ 2.76 (1H, td, J=5.6, 2.6 Hz, H-4), 2.20 (3H, 5, H-19), 1.99 (1H, m, H-13A), 1.90 (1H, m, H-13B), 1.90 (1H, m, H-3A), 1.86 (1H, m, H-2A), 1.83 (1H, m, H-11), 1.68 (3H, 5, H-16), 1.66 (1H, m, H-2B), 1.58 (3H, 5, H-17), 1.51 (1H, m, H-3B), 1.25 (1H, m, H-12A), 1.36 (2H, m, H-12B), 1.21 (3H, d, J=7.0 Hz, H-20). ¹³C NMR (100 MHz, CDCl₃) δ: 15.54 (C-19), 17.68 (C-17), 20.51 (C-2), 21.11 (C-20), 25.70 (C-16), 26.16 (C-13), 26.47 (C-3), 26.98 (C-1), 29.85 (C-12), 37.91 (C-4), 45.45 (C-11), 64.38 (C-18), 121.31 (C-6), 122.00 (C-5), 124.43 (C-14), 127.18 (C-9), 130.70 (C-10), 131.75 (C-15), 139.74 (C-7), 141.61 (C-8).

5,18-epoxyserrulat-14-en-8,18-diol (2)

Red liquid, yield 84 mg, [α]_(D) ²⁰ −21.5° (C=1.0, CHCl₃). UV (CH₃OH) λ_(max) nm (log ε) 293 (3.36) and 334 (2.91). IR (v): 3327, 2970, 2926, 2860, 1647, 1558, 1448, 1043 cm⁻¹. HRESIMS: 315.1970 m/z [M−H]⁻ (calcd 315.1966). C₂₀H₂₈O₃. ¹H NMR (400 MHz, CDCl₃) δ 6.48 (1H, 5, H-7), 5.59 (1H, bs, H-18), 5.14 (1H, bt, J=7.0 Hz, H-14), 3.11 (1H, sextet, J=7.6, H-1), 2.54 (1H, td, J=11.4, 3.6 Hz, H-4), 2.25 (1H, m, H-2A), 2.20 (1H, m, H-13A), 2.13 (3H, 5, H-19), 2.09 (1H, m, H-13B), 2.08 (1H, m, H-3A), 1.69 (1H, m, H-12A), 1.70 (3H, 5, H-16), 1.63 (3H, s, H-17), 1.52 (1H, m, H-11), 1.41 (1H, m, H-2B), 1.28 (3H, d, J=6.9 Hz, H-20), 1.27 (1H, m, H-12B), 1.05 (1H, m, H-3B). ¹³C NMR (100 MHz, CDCl₃) δ 15.88 (C-19), 17.75 (C-17), 22.89 (C-20), 25.36 (C-13), 25.70 (C-16), 26.62 (C-3), 27.79 (C-1), 28.90 (C-12), 31.75 (C-2), 31.99 (C-4), 40.48 (C-11), 91.88 (C-18), 115.94 (C-7), 122.94 (C-6), 124.09 (C-14), 124.29 (C-10), 125.21 (C-9), 131.99 (C-15), 141.71 (C-5), 146.85 (C-8).

5,18-epoxy-8-hydroxyserrulat-14-ene (3)

Shiny yellow liquid, yield 87 mg, [α]_(D) ²⁰ −8.9° (C=1.0, CHCl₃). UV (CH₃OH) λ_(max) nm (log ε) 296 (3.55) and 334 (2.66). IR (v): 3502, 2922, 2856, 1622, 1597, 1421, 1230 cm⁻¹. HRESIMS: 301.21635 m/z [M+H]⁺ (calcd 301.21621) C₂₀H₂₈O₂. ¹H NMR (400 MHz, CDCl₃) δ: 6.46 (1H, 5, H-7), 5.13 (1H, bt, J=6.9 Hz, H-14), 4.35 (1H, dd, J=10.5, 3.7 Hz, H-18A), 3.70 (1H, t, J=10.8 Hz, H-18B), 3.13 (1H, sextet, J=7.4 Hz, H-1), 2.31 (1H, m, H-4), 2.24 (1H, m, H-2A), 2.16 (1H, m, H-13A), 2.15 (1H, m, H-3A), 2.12 (3H, s, H-19), 2.02 (1H, m, H-13B), 1.74 (1H, m, H-12A), 1.70 (3H, 5, H-16), 1.63 (3H, 5, H-17), 1.55, (1H, m, H-11), 1.40 (1H, m, H-2B), 1.28 (3H, d, J=7.0 Hz, H-20), 1.18 (1H, m, H-12B), 1.06 (1H, td J=12.2, 4.4 Hz, H-3B). ¹³C NMR (100 MHz, CDCl₃) δ: 15.82 (C-19), 17.73 (C-17), 23.12 (C-20), 25.44 (C-13), 25.70 (C-16), 26.97 (C-3), 27.61 (C-1), 29.97 (C-12), 31.47 (C-2), 37.17 (C-11), 39.00 (C-4), 69.52 (C-18), 115.79 (C-7), 122.64 (C-6), 124.12 (C-14), 124.26 (C-9), 125.70 (C-10), 131.99 (C-15), 145.27 (C-5), 146.16 (C-8).

7,8-dihydroxyserrulat-14-ene (4)

Yellowish liquid, yield 56 mg, [α]_(D) ²⁰ −21.1° (C=1.0, CHCl₃). UV (CH₃OH) λ_(max) nm (log ε) 278 (3.36) and 329 (2.95). IR (v): 3396, 2954, 2924, 2868, 1635, 1577, 1456 cm⁻¹. HRESIMS: 325.21413 m/z [M+Na]⁺ (calcd 325.21380) C₂₀H₃₀O₂. ¹H NMR (400 MHz, CDCl₃) δ 6.53 (1H, 5, H-5), 5.00 (1H, bt, J=7.0 Hz, H-14), 3.06 (1H, pentet of d, J=6.7, 2.8 Hz, H-1), 2.53 (1H, td, J=5.5, 3.4 Hz, H-4), 2.22 (3H, 5, H-19), 2.00 (1H, m, H-13A), 1.95 (1H, m, H-2A), 1.84 (1H, m, H-11), 1.82 (1H, m, H-3A), 1.80 (1H, m, H-13B), 1.70 (1H, m, H-3B), 1.67 (3H, 5, H-16), 1.59 (3H, 5, H-17), 1.48 (1H, m, H-2B), 1.29 (1H, m, H-12A), 1.12 (3H, d, J=6.8 Hz, H-20), 1.05 (1H, m, H-12B), 0.95 (3H, d, J=6.8, H-18). ¹³C NMR (100 MHz, CDCl₃) δ: 15.53 (C-19), 17.62 (C-17), 18.66 (C-18), 19.63 (C-3), 21.10 (C-20), 25.71 (C-16), 26.25 (C-13), 27.1 (C-1), 27.43 (C-2), 33.35 (C-12), δ 37.8 (C-11), δ 41.88 (C-4), δ 120.50 (C-6), δ 122.46 (C-5), δ 124.96 (C-14), δ 127.09 (C-9), δ 131.10 (C-15), δ 131.90 (C-10), δ 139.19 (C-7), δ 140.93 (C-8).

Serrulat-14-en-5,8-dione (5)

Light orange liquid, yield 30 mg. ¹H NMR (400 MHz, CDCl₃) δ 6.68 (1H, 5, H-5), 5.02 (1H, bt, J=7.1 Hz, H-14), 2.85 (1H, m, H-1), 2.20 (1H, m, H-4), 2.12 (1H, m, H-3A), 2.03 (1H, m, H-13A), 1.95 (3H, 5, H-19), 1.88 (1H, m, H-11), 1.85 (1H, m, H-13B), 1.69 (1H, m, H-2A), 1.67 (3H, 5, H-16), 1.58 (3H, 5, H-17), 1.34 (1H, m, H-2B), 1.30 (1H, m, H-12A), 1.20 (1H, m, H-3B), 1.12 (1H, m, H-12B), 1.07 (6H, d, J=6.9 Hz, H-18 and H-20). ¹³C NMR (100 MHz, CDCl₃) δ 15.21 (C-19), 17.69 (C-17), 18.67 (C-20), 18.75 (C-18), 20.55 (C-3), 25.71 (C-16), 26.10 (C-13), 26.56 (C-1), 26.59 (C-2), 33.41 (C-12), 34.9 (C-11), 43.83 (C-4), 124.13 (C-14), 132.00 (C-15), 135.36 (C-6), 140.04 (C-7), 140.70 (C-9), 150.13 (C-10), 179.85 (C-5), 181.55 (C-8).

5,18-epoxyserrulat-14-en-7,8-dione (6)

Purple liquid, yield 50 mg, HRESIMS: 315.19573 m/z [M+H]+(calcd 315.19547) C₂₀H₂₆O₃. ¹H NMR (400 MHz, CDCl₃) δ 5.07 (1H, bt, J=6.8 Hz, H-14), 4.49 (1H, dd, J=11.0, 3.8 Hz, H-18A), 3.86 (1H, t, J=11.1 Hz, H-18B), 2.82 (1H, m, H-1), 2.14 (1H, m, H-4), 2.15 (1H, m, H-2A), 2.13 (1H, m, H-3A), 2.09 (1H, m, H-13A), 1.97 (1H, m, H-13B), 1.81 (3H, 5, H-19), 1.76 (1H, m, H-12A), 1.74 (1H, m, H-11), 1.71 (3H, 5, H-16), 1.63 (3H, 5, H-17), 1.23 (1H, m, H-2B), 1.19 (1H, m, H-12B), 1.13 (3H, d, H-20) 1.11 (1H, m, H-3B). ¹³C NMR (100 MHz, CDCl₃) δ 7.57 (C-19), 17.78 (C-17), 21.67 (C-20), 24.66 (C-13), 25.32 (C-3), 25.71 (C-16), 28.74 (C-1), 29.15 (C-12), 30.77 (C-2), 37.07 (C-11), 40.09 (C-4), 71.85 (C-18), 114.44 (C-6), 123.06 (C-14), 132.87 (C-15), 139.43 (C-9), 149.67 (C-10), 162.76 (C-5), 179.38 (C-7), 181.91 (C-8).

Compound 6 was observed to be formed from oxidation of Compound 1.

Acetylation of Serrulatane Diterpenes

7,8,18-triacetyloxyserrulat-14-ene (7)

The serrulatane (1) (400 mg) was dissolved in acetic anhydride (1 mL) and in dry pyridine (1 mL) and stirred at room temperature overnight (Davis et al., 1999). The reaction mixture was quenched with distilled water (30 mL), and was extracted with dichloromethane (3×40 mL). The dichloromethane solution was extracted with aqueous 0.1 M hydrochloric acid (30 mL) to remove the residual trace of pyridine. The dichloromethane solutions were dried with Na₂SO₄, filtered and concentrated on a rotary evaporator to give a yellow oily residue. Finally, using normal-phase short-column vacuum chromatography (NPSCVC) with dichloromethane and ethyl acetate (100:0 to 90:10, v/v) the compound (7) (434 mg, 77%) was purified and isolated as a pale yellow oil, yield 27 mg. HRESIMS: 467.2404 m/z [M+Na]⁺ (calcd 467.2206). C₂₆H₃₆O₆. ¹H NMR (400 MHz, CDCl₃) δ 6.92 (1H, 5, H-5), 4.97 (1H, bt, J=7.1 Hz, H-14), 4.11 (2H, d, J=6.3 Hz, H-18), 2.92 (1H, m, H-1), 2.88 (1H, m, H-4), 2.31 (3H, 5, H-26), 2.29 (3H, s, H-24), 2.14 (3H, 5, H-19), 2.12 (1H, m, H-11), 2.06 (3H, 5, H-22), 1.99 (1H, m, H-13A), 1.87 (1H, m, H-13B), 1.87 (1H, m, H-3A), 1.87 (1H, m, H-2A), 1.68 (1H, m, H-3B), 1.66 (3H, 5, H-16), 1.54 (3H, 5, H-17), 1.50 (1H, m, H-2B), 1.33 (1H, m, H-12A), 1.23 (1H, m, H-12B), 1.15 (3H, d, J=7.0 Hz, H-20). ¹³C NMR (100 MHz, CDCl₃) δ 16.08 (C-19), 17.60 (C-17), 19.16 (C-3), 20.36 (C-24), 20.51 (C-26), 20.97 (C-22), 21.45 (C-20), 25.68 (C-16), 26.11 (C-13), 26.96 (C-2), 27.58 (C-1), 28.32 (C-12), 36.92 (C-4), 41.92 (C-11), 65.88 (C-18), 123.94 (C-14), 128.09 (C-5), 128.31 (C-6), 132.04 (C-15), 139.26 (C-7), 140.56 (C-8), 134.06 (C-9), 137.11 (C-10), 168.1 (C-23), 168.34 (C-25), 171.18 (C-21).

5,18-epoxyserrulat-14-ene-8,18-diacetate (8)

To acetylate compound (2), 80 mg of serrulatane (2) was dissolved in acetic anhydride (1 mL) and in dry pyridine (1 mL) and stirred at room temperature overnight, and with the same workup procedure and using short-column vacuum chromatography with hexane and ethyl acetate (100:0 to 80:20, v/v) the compound (8) (57.8 mg, 58%) was purified and isolated. Red coloured liquid, yield 18 mg, HRESIMS: m/z 423.2143 [M+Na]⁺ calcd 423.2142. C₂₄H₃₂O₅. ¹H NMR (400 MHz, CDCl₃) δ: 6.69 (1H, 5, H-7), 6.53 (1H, d, J=1.7 Hz, H-18), 5.08 (1H, bt, J=6.5 Hz, H-14), 2.96 (1H, sextet, J=7.4 Hz, H-1), 2.54 (1H, td, J=11.6, 3.8 Hz, H-4), 2.3 (3H, 5, H-24), 2.19 (1H, m, H-13A), 2.19 (1H, m, H-2A), 2.14 (3H, 5, H-19), 2.12 (1H, m, H-3A), 2.09 (3H, 5, H-22), 2.02, (1H, m, H-13B), 1.70 (1H, m, H-12A), 1.69 (3H, 5, H-16), 1.62 (3H, 5, H-17), 1.65 (1H, m, H-11), 1.42 (1H, m, H-2B), 1.30 (1H, m, H-12B), 1.23 (3H, d, J=6.8 Hz, H-12), 1.10 (1H, m, H-3B). ¹³C NMR (100 MHz, CDCl₃) δ: 15.88 (C-19), 17.68 (C-17), 21.09 (C-22), 21.23 (C-24), 22.78 (C-20), 25.15 (C-13), 25.70 (C-16), 26.22 (C-3), 28.26 (C-1), 28.29 (C-12), 31.41 (C-2), 32.3 (C-4), 38.93 (C-11), 90.18 (C-18), 41.92 (C-11), 122.53 (C-7), 123.48 (C-14), 123.77 (C-10), 123.96 (C-6), 130.9 (C-9), 132.57 (C-15), 142.44 (C-8), 145.63 (C-5), 170.05 (C-23), 170.19 (C-21).

Methylation of Serrulatane Diterpenes

8-methoxy-7,18-dihydroxyserrulat-14-ene (11) and 7-methoxy-8,18-dihydroxyserrulat-14-ene (12)

Compound 1 (140 mg) was treated with diazomethane (excess equiv) in diethyl ether at 0° C. to room temperature overnight to give an undetermined 4 to 1 (¹H-NMR) mixture of mono-methylated products 11 and 12 (35 mg, 88% purity by HPLC).

Compounds 11 and 12

Yellow oil Mass Spectrum (positive mode) m/z=333.2 (12%, (M+H)⁺), 315.2 (100%, (M+H—H₂O)⁺), 205.2 (18%, (M+H—C₈H₁₆O)⁺). HPLC analysis: Wavelength, 210 nm, bandwidth 4; Column, SorbTech C18AQ, 2.1×50 mm, 3 μm; Retention time 7.474 min; Mobile phase acetonitrile/formic acid/water; Gradient method, 5-95% CAN+0.1% Formic acid in Water+0.1% Formic acid in 14 min, hold at 95% CAN+Formic acid for 4 min. U_(Vmax), 275 nm and 210 nm, shoulder at 225 nm. ¹H-NMR analysis indicated a 4:1 undetermined mixture of 11 and 12 (interchangeable major and minor isomers). ¹H NMR (400 MHz, CDCl₃) δ: 6.74 (1H, 5, H-5)[minor], 6.56, (1H, 5, H-5)[major], 5.78 (1H, OH)[major], 5.57 (1H, OH)[minor], 5.01 (1H, bt, J=6.3 Hz, H-14), 3.78 (3H, 5, CH₃O)[minor], 3.77 (3H, 5, CH₃O)[major], 3.63 (2H, m, H-18), 3.14 (1H, m, H-1)[major], 3.07 (1H, m, H-1)[minor], δ 2.81 (1H, m, H-4), 2.25 (3H, 5, H-19)[major], 2.21 (3H, 5, H-19)[minor], 2.0-1.8 (5H, m, H-13, H-3A, H-2A, H-11), 1.7-1.6 (1H, m, H-2B), 1.66 (3H, bs, H-16), 1.55 (3H, bs, H-17), 1.52-1.46 (1H, m, H-3B), 1.35-1.23 (2H, m, H-12), 1.21 (3H, d, J=6.8 Hz, H-20)[major] 1.20 (3H, d, J=6.8 Hz, H-20)[minor], ¹³C NMR (100 MHz, CDCl₃) δ: 15.50 (C-19)[minor], 15.77 (C-19)[major], 17.62 (C-17), 20.22 (C-2)[major], 20.44 (C-2)[minor], 21.08 (C-20)[major], 22.22 (C-20)[minor], 25.66 (C-16), 26.27 (C-13), 26.81 (C-3), 27.14 (C-1)[major], 27.87 (C-1)[minor], 29.14 (C-12)[minor], 29.26 (C-12)[major], 37.53 (C-4)[minor], 37.55 (C-4)[major], 45.43 (C-11)[minor], 45.53 (C-11)[major], 60.64 (OCH₃)[major], 60.89 (OCH₃)[minor], 64.17 (C-18)[major], 64.30 (C-18)[minor], 121.89 (C-5), 124.55 (C-14), 126.39, 126.70, 127.69, 130.42, 131.63, 131.66, 133.17, 135.11, 143.15, 144.85, 145.26, 146.27.

FIG. 1 and FIG. 2 show the ¹H NMR (400 MHz, CDCl₃) and the ¹³C NMR (100 MHz, CDCl₃) spectra of the 4:1 mixture of compounds 11 and 12.

Biological Evaluation of Serrulatane Terpene Compounds

The serrulatane diterpenes 1 to 3 were evaluated for in vitro biological activities using assays for epigenetics (Tables 3 to 12). Table 3 shows the experimental conditions used in epigenetic binding assays. Table 4 shows the results of inhibition of compounds 1 to 3 on binding of epigenetic interacting protein modules. Tables 5 to 8 show representative inhibition data for Compound 1 on binding example epigenetic targets (Table 5: ASH1L (h) bromodomain; Table 6: CECR2(h) bromodomain; Table 7: SP140 (h) bromodomain; Table 8: UHRF1(108-286) (h)). Corresponding binding curves for the data displayed in Tables 5 to 8 are shown in FIGS. 3 to 6. Table 9 shows IC₅₀ values from inhibition/concentration-response curves of compounds 1 and 2 for binding of epigenetic interacting protein modules against reference compounds.

Table 10 shows the inhibition of compounds 1 to 3 at 10 μM on activity of epigenetic enzymes against references.

Table 11 shows IC₅₀ values from inhibition/concentration-response curves of compounds 1, 2 and 4 for inhibition of epigenetic enzymes against reference compounds. Table 12 shows the pharmacological activity of compounds 1 to 3. Diterpenes 1 to 3 were also evaluated for in vitro pharmacological activity (Tables 13 to 16). Table 13 shows pharmacology assay methods. Table 14 shows pharmacological activity of compounds 1 to 3 at 10 μM. Table 15 shows activity of Compound 1 against 5-lipoxygenase, lipid peroxidase and monoamine oxidase (MAO-A) IC₅₀ (μM) and reference compounds. Table 16 shows the effect of compounds 1 to 4 at 10 μM on 2-deoxy-D-glucose uptake into muscle cells.

Table 17 shows dose response and IC₅₀ values for the pharmacological activity of representative serrulatane diterpenes.

Diterpenes 1 to 4 and Myoporum insulare resin were evaluated in a cell viability assay (Table 18)

Diterpenes 1, 2, 4 and Myoporum insulare resin were assayed for cell cancer cell proliferation in an OncoPanel assay (Table 19 to 29). Table 21 shows Oncopanel cell proliferation results for Compounds 11 and 12.

Epigenetic Assays

Epigenetic binding assays were carried out by Eurofins CEREP (France). The conditions used in the assays including: enzymes, source of enzymes, substrate, substrate concentration, ligand, ligand concentration, tracer, incubation, detection method, and reference compound and concentration are detailed in Table 3. Appropriate literature references are also provided. The AlphaScreen detection method refers to an Amplified Luminescent Proximity Homogeneous Assay used to study biomolecular interactions (see Ullman el al. 1994). LANCE and LanthaScreen assays are based on the principle of time-resolved fluorescence energy transfer (TR-FRET) and are described, for example, in Ma et al. 2008.

The IC₅₀ values (concentration causing a half-maximal inhibition of control specific activity) were determined by non-linear regression analysis of the inhibition/concentration-response curves generated with mean replicate values using Hill equation curve fitting. Inhibition/concentration-response curve concentrations were 30, 3, 0.3, 0.03 and 0.003 μM.

TABLE 3 Epigenetic binding assay details Assay Main Feature Bromodomain ATAD2B bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 Ligand concentration 75 nM Incubation 30 min/RT Detection method AlphaScreen Reference: Ischemin sodium salt (IC50: 76.6 μM) ASH1L bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H4 di acetyl Lys 5/8 Ligand concentration 75 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 13.8 μM) BAZ2A bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 25 nM Incubation 30 min/RT Detection method AlphaScreen Reference: G5K2801 (IC50: 1.8 μM) BRPF1-1 bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 Ligand concentration 10 nM Incubation 30 min/RT Detection method AlphaScreen Reference: Bromosporine (IC50: 0.026 μM) CECR2 bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 Ligand concentration 10 nM Incubation 30 min/RT Detection method AlphaScreen Reference: Bromosporine (IC50: 0.062 μM) EP300 bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H3 acetyl Lys56 2012] Ligand concentration 25 nM Incubation 30 min/RT Detection method AlphaScreen Reference: SGC-CBP30 (IC50: 0.050 μM) KAT2A (GCN5L2) bromodo Source human recombinant (E. coli) main [Filippakopoulos et al. Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 30 nM Incubation 15 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 45 μM) PCAF bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 100 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 12 μM) PB1(2) bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H3 acetyl Lys 14 Ligand concentration 60 nM Incubation 15 min/RT Detection method AlphaScreen Reference: PFI-3 (IC50: 68 μM) PB1(3) bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H3 acetyl Lys14 Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 47 μM) PB1(4) bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H3 acetyl Lys14 Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: Ischemin sodium salt (IC50: 6.8 μM) PHIP(2) bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H4 diacetyl Lys5/8 Ligand concentration 100 nM Incubation 30 min/RT Detection method AlphaScreen Reference: SGC-CBP30 (IC50: 0.130 μM) SP140 bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand biotin-H3 acetyl Lys 9 Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: Ischemin (IC50: 86 μM) SMARCA2 bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand Biotin H3 mono acetyl Lys 14 Ligand concentration 5 nM Incubation 30 min/RT Detection method AlphaScreen Reference: PFI-3 (IC50: 0.47 μM) TAF1(1) bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 100 nM Incubation 30 min/RT Detection method AlphaScreen Reference: G5K2801 (IC50: 4.4 μM) SMARCA4 bromodomain Source human recombinant (E. coli) [Filippakopoulos et al. 2012] Ligand Biotin-H3 mono acetyl Lys14 Ligand concentration 15 nM Incubation 30 min/RT Detection method AlphaScreen Reference: PFI-3 (IC50: 3 μM) TAF1(2) bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 100 nM Incubation 30 min/RT Detection method AlphaScreen Reference: G5K2801 (IC50: 10 μM) BAZ2B bromodomain Source human recombinant (E. coli) [Philpott et al. 2011] Ligand biotin-H3 acetyl Lys14 Ligand concentration 25 nM Incubation 30 min/RT Detection method AlphaScreen Reference: G5K2801 (IC50: 1.8 μM) ATAD2A bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 100 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 70 μM) BRD2(1) bromodomain Source human recombinant (E. coli) [Philpott et al. 2011] Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-11 (IC50: 0.510 μM) BRD2(2) bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 0.017 μM) BRD3(1) bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 25 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 0.040 μM) BRD3(2) bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 di acetyl Lys 5/8 2012] Ligand concentration 100 nM Incubation 30 min/RT Detection method AlphaScreen Reference: PFI-1 (IC50: 0.79 μM) BRD4(1) bromodomain Source human recombinant (E. coli) [Philpott et al. 2011] Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 0.018 μM) BRD4(2) bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: PFI-1 (IC50: 1.3 μM) BRDT(1) bromodomain Source human recombinant (E. coli) [Filippakopoulos and Knapp Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 2012] Ligand concentration 25 nM Incubation 30 min/RT Detection method AlphaScreen Reference: JQ-1 (IC50: 0.110 μM) CREBBP bromodomain Source human recombinant (E. coli) [Philpott et al. 2011] Ligand biotin-H3 acetyl Lys56 Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: SGC-CBP30 (IC50: 0.120 μM) FALZ bromodomain Source human recombinant (E. coli) [Philpott et al. 2011] Ligand biotin-H4 tetra acetyl Lys 5/8/12/16 Ligand concentration 50 nM Incubation 30 min/RT Detection method AlphaScreen Reference: I-CBP112 (IC50: 24.5 μM) Chromodomain CBX1 [Kaustov et al. 2011] Source human recombinant (E. coli) Ligand biotin-H3 trimethylated Lys9 Ligand concentration 5 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA CBX2 [Kaustov et al. 2011] Source human recombinant (E. coli) Ligand biotin-H3 trimethylated Lys27 Ligand concentration 20 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA CBX4 [Kaustov et al. 2011] Source human recombinant (E. coli) Ligand biotin-H3 trimethylated Lys27 Ligand concentration 8 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA CBX6 [Kaustov et al. 2011] Source human recombinant (E. coli) Ligand biotin-H3 trimethylated Lys27 Ligand concentration 5 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA CBX5 [Kaustov et al. 2011] Source human recombinant (E. coli) Ligand biotin-H3 trimethylated Lys9 Ligand concentration 6 nM Incubation 15 min/RT Detection method AlphaScreen Reference: NA CBX7 [Kaustov et al. 2011] Source human recombinant (E. coli) Ligand biotin-H3 trimethylated Lys27 Ligand concentration 10 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA CBX8 [Kaustov et al. 2011] Source human recombinant (E. coli) Ligand biotin-H3 trimethylated Lys27 Ligand concentration 55 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA MBT domain L3MBTL1 [Kim et al. 2006] Source human recombinant (E. coli) Ligand biotin- H3 methylated Lys4 Ligand concentration 50 nM Incubation 15 min/RT Detection method AlphaScreen Reference: NA L3MBTL3 [Kim et al. 2006] Source human recombinant (E. coli) Ligand biotin-H4 dimethylated lysine 20 Ligand concentration 60 nM Incubation 15 min/RT Detection method AlphaScreen Reference: NA PHD Domain SP140 [Org et al. 2008] Source human recombinant (E. coli) Ligand Biotin H3K4me Ligand concentration 15 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA TRIM 33 [Venturini et al. Source human recombinant (E. coli) 1999] Ligand biotin-H3 trimethylated Lys9 Ligand concentration 8 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA UHRF1(108-286) [Xie et al. Source human recombinant (E. coli) 2012] Ligand biotin-H3 trimethylated Lys9 Ligand concentration 15 nM Incubation 15 min/RT Detection method AlphaScreen Reference: NA PHF20(1) [Adams-Cioba et al. 2012] Source human recombinant (E. coli) Ligand biotin-H4 dimethylated Lys20 Ligand concentration 100 nM Incubation 30 min/RT Detection method AlphaScreen Reference: NA Cell-based detection methyl modifications H3K27 ac Source HeLa Cells [Hayashi-Takanaka Incubation 24 h/37° C. et al. 2011] Detection method AlphaScreen Reference: NA H3K27 me2-1 [Kubicek, S. Source MCF7 cells et al. 2007] Incubation 24 h/37° C. Detection method AlphaScreen Reference: NA H3K27 me3 [Kubicek, S. Source SU-DHL-6 cells et al. 2007] Incubation 24 h/37° C. Detection method AlphaScreen Reference: NA H3K36 me2 [Kubicek, S. Source HeLa cells et al. 2007] Incubation 24 h/37° C. Detection method AlphaScreen Reference: NA H3K4 me2 [Ken-ichi Noma Source HeLa cells and Grewal 2002] Incubation 24 h/37° C. Detection method AlphaScreen Reference: NA H3K79 me2 [Kubicek, S. Source MCF7 cells et al. 2007] Incubation 24 h/37° C. Detection method AlphaScreen Reference: NA H3K9 ac [Hayashi-Takanaka Source HeLa cells et al. 2011] Incubation 24 h/37° C. Detection method AlphaScreen Reference: NA H3K9 me2 [Kubicek, S. Source HeLa cells et al. 2007] Incubation 24 h/37° C. Detection method AlphaScreen Reference: NA Demethylases (KDMs) FBXL10 (h) [Rotili and Mai Source human recombinant (Sf9 cells) 2011] Substrate biotin-H3K36me2 (24 nM) Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.61 μM) FBXL11 (h) [Chowdhury Source human recombinant (E. coli) 2011] Ligand biotin- H3K36me2 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 1.5 μM) JARID1A (h) [Nottke et al. Source human recombinant (Sf9 cells) 2009] Ligand biotin-H3K4me3 Ligand concentration 100 nM Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.18 μM) JARID1B (h) [Kristensen Source human recombinant (Sf9 cells) 2012] Ligand biotin-H3K4me3 Ligand concentration 60 nM Incubation 30 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.15 μM) JARID1C (h) [Nottke et al. Source human recombinant (Sf9 cells) 2009] Substrate biotin-H3K4me3 Substrate concentration 15 nM Incubation 30 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.23 μM) JMJD1A (h) [Heightman Source human recombinant (E. coli) 2011] Substrate biotin-H3K4me1 Substrate concentration 25 nM Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.76 μM) JMJD2A (h) [King et al. Source human recombinant (E. coli) 2010] Ligand biotin-H3K9me3 Ligand concentration 100 nM Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.083 μM) JMJD2B (h) [King et al. Source human recombinant (E. coli) 2010] Ligand biotin-H3K9me3 Ligand concentration 100 nM Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.14 μM) JMJD2C (h) [King et al. Source human recombinant (E. coli) 2010] Ligand biotin-H3K9me3 Ligand concentration 100 nM Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.091 μM) JMJD2D (h) [Hong et al. Source human recombinant (Sf9 cells) 2007] Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.091 μM) JMJD2E (h) [Hong et al. Source human recombinant (Sf9 cells) 2007] Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE Reference: 2,4 PDCA (IC50: 0.061 μM) JMJD3 (h) [Hong et al. 2007] Source human recombinant (Sf9 cells) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE 1198 Reference: 2,4 PDCA (IC50: 52 μM) LSD1 (h) [Hong et al. 2007] Source human recombinant (Sf9 cells) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE Reference: Tranylcypromine (IC50: 22 μM) PHF8(h) [Hong et al. 2007] Source human recombinant (Sf9 cells) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE 1198 Reference: Daminozide (IC50: 0.28 μM) UTX (h) [Hong et al. 2007] Source human recombinant (Sf9 cells) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE Reference: IOX1 (0.15 μM) DNA methyltransferases (DNMTs) DNMT1 (h) [Pradhan et al. Source human recombinant (E. coli) 1999] Substrate Poly (dI-dC)-Poly(dIdC) [6 mU/mL] Tracer [3H] SAM (50 nM) Incubation 30 min/37° C. Detection method Scintillation counting Reference: SAH (IC50: 0.23 μM) DNMT3b [Hong et al. 2007] Source human recombinant (Sf9 cells) Ligand biotin- H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE Reference: SAH (IC50: 0.094 μM) DNMT3B/DNMT3L (h) Source human recombinant (Sf9 cells) [Suetake et al. 2004] Substrate Poly (dI-dC)-Poly(dIdC) [0.15 mU/mL] Tracer [3H] SAM (200 nM) Incubation 20 min/37° C. Detection method Scintillation counting Reference: SAH (IC50: 0.045 μM) hDNMT3a [Aoki et al. 2001] Source human recombinant (Sf9 cells) Substrate Poly (dI-dC)-Poly(dIdC) Tracer [3H] SAM (200 nM) Incubation 20 min/37° C. Detection method Scintillation counting Reference: SAH (IC50: 0.052 μM) Histone acetyl-transferases (HATs) CREBBP(h) [Hong et al. 2007] Source human recombinant (E. coli) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE Reference: Garcinol (IC50: 1.5 μM) GCN5L2(h) [Hong et al. 2007] Source human recombinant (Sf9 cells) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method LANCE Reference: Anacardic acid (IC50: 4.7 μM) HAT1(h) [Zhang et al. 2012] Source human recombinant (E. coli) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method Scintillation counting Reference: Garcinol (IC50: 1.5 μM) MYST3(h) [Zhang et al. 2012] Source human recombinant (E. coli) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method Scintillation counting Reference: Garcinol (IC50: 1.8 μM) MYST4 (h) [Zhang et al. 2012] Source human recombinant (E. coli) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method Scintillation counting 1293 Reference: Curcumin (IC50: 11 μM) pCAF(h) [Zhang et al. 2012] Source human recombinant (E. coli) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method Scintillation counting 1293 Reference: Garcinol (IC50: 2.9 μM) TIP60 (h) [Zhang et al. 2012] Source human recombinant (Sf 21 cells) Ligand biotin-H3K27me3 Ligand concentration 50 nM Incubation 10 min/RT Detection method Scintillation counting 1293 Reference: Garcinol (IC50: 1.5 μM) Histone deacetylase (HDACs) [Strahl and Allis 2000] HDAC1 (h) Source human recombinant Substrate fluorogenic HDAC substrate Substrate concentration 20 μM Incubation 10 min/RT Detection method fluoro-lysine fluorimetry Reference: trichostatin A (IC50: 0.0052 μM) HDAC2 (h) Source human recombinant fluorogenic HDAC substrate Ligand NA Substrate concentration 20 μM Incubation 15 min/RT Detection method fluoro-lysine fluorimetry 896 Reference: trichostatin A (IC50: 0.024 μM) HDAC3 (h) Source human recombinant Substrate fluorogenic HDAC substrate Substrate concentration 50 μM Incubation 10 min/RT Detection method fluoro-lysine fluorimetry Reference: trichostatin A (IC50: 0.0068 μM) HDAC4 (h) Source human recombinant Substrate fluorogenic HDAC substrate Substrate concentration 20 μM Incubation 30 min/RT Detection method fluoro-lysine fluorimetry Reference: trichostatin A (IC50: 4.0 μM) HDAC5 (h) Source human recombinant Ligand fluorogenic HDAC substrate Substrate concentration 20 μM Incubation 30 min/RT Detection method fluoro-lysine fluorimetry Reference: trichostatin A (IC50: 1.0 μM) HDAC6 (h) Source human recombinant Substrate fluorogenic HDAC substrate Substrate concentration 25 μM Incubation 30 min/RT Detection method fluoro-lysine fluorimetry Reference: trichostatin A (IC50: 0.0074 μM) HDAC7 (h) Source human recombinant Substrate fluorogenic HDAC substrate class 2a Substrate concentration 50 μM Incubation 45 min/RT Detection method fluoro-lysine fluorimetry Reference: trichostatin A (IC50: 1.8 μM) HDAC8 (h) Source human recombinant Substrate fluorogenic HDAC substrate class 2a Substrate concentration 50 μM Incubation 45 min/RT Detection method fluoro-lysine fluorimetry 896 Reference: trichostatin A (IC50: 0.41 μM) HDAC9 (h) Source human recombinant Substrate fluorogenic HDAC substrate class 2a Substrate concentration 50 μM Incubation 30 min/RT Detection method fluoro-lysine fluorimetry 896 Reference: trichostatin A (IC50: 9.1 μM) HDAC10 (h) Source human recombinant Substrate fluorogenic HDAC substrate class 2a Substrate concentration 50 μM Incubation 45 min/RT Detection method fluoro-lysine fluorimetry 896 Reference: trichostatin A (IC50: 0.009 μM) HDAC11 (h) Source human recombinant (E. coli) Substrate fluorogenic HDAC substrate class 2a Substrate concentration 50 μM Incubation 30 min/37° C. Detection method fluoro-lysine fluorimetry 896 Reference: Scriptaid (IC50: 8.9 μM) Sirtuins sirtuin 1 (h) (inhibitor effect) Source human recombinant (E. coli) [Strahl and Allis 2000] Substrate fluorogenic HDAC substrate class 2a Substrate concentration 50 μM Incubation 30 min/37° C. Detection method fluoro-lysine fluorimetry Reference: Suramin (IC50: 7.9 μM) sirtuin 2 (h) (inhibitor effect) Source human recombinant (E. coli) [Michan and Sinclair 2007] Substrate fluoro-lysine sirtuin 2 deacetylase substrate Substrate concentration 150 μM Incubation 60 min/RT Detection method fluoro-lysine fluorimetry Reference: Suramin (IC50: 21 μM) sirtuin 3 (h) (inhibitor effect) Source human recombinant (E. coli) [Strahl and Allis 2000] Substrate fluorogenic HDAC substrate class 2a Substrate concentration 50 μM Incubation 30 min/37° C. Detection method fluoro-lysine fluorimetry Reference: Niacinamide (IC50: 21 μM) Sirtuin 6 (h) Source human recombinant (E. coli) [Michishita et al. Substrate Fluorogenic HDAC 2008] Substrate concentration 50 μM Incubation 180 min/RT Detection method fluoro-lysine fluorimetry Reference EX-527 (IC50: 550 μM) Sirtuin 7 (h) [Kim and Kim Source human recombinant (Sf9 cells) 2013] Substrate fluorogenic HDAC Substrate concentration 25 μM Incubation 60 min/37° C. Detection method fluoro-lysine fluorimetry Reference: JFD00244 (IC50: 1300 μM) Methyltransferases (MTs) ASH1L [An et al. 2011] Source human recombinant (E. coli) Substrate polynucleosome (1.5 μg/mL) Tracer [3H] SAM (150 nM) Incubation 15 min/RT Detection method Scintillation counting Reference: SAH (IC50: 9.1 μM) DOT1L (h) [An et al. 2011] Source human recombinant (E. coli) Substrate polynucleosome (2.5 μg/mL) Tracer [3H] SAM (150 nM) Incubation 15 min/RT Detection method Scintillation counting Reference: SAH (IC50: 0.14 μM) EHMT1 (h) [Yost et al. 2011] Source human recombinant (E. coli) Substrate (concentration) histone H3 full length (10 nM) Co-substrate (concentration) [3H] SAM (25 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: SAH (IC50: 0.18 μM) EZH1/EED/SUZ12 (h) [Shen Source human recombinant et al. 2008] Substrate (concentration) histone H3 full length (70 nM) Co-substrate (concentration) [3H] SAM (120 nM) Incubation 90 min/RT Detection method Scintillation counting Reference: SAH (IC50: 8.4 μM) EZH2/EED/SUZ12 (h) Source human recombinant (Sf9 cells) (PRC2 complex) [Philpott et Substrate (concentration) histone H3 full length (50 nM) al. 2011] Co-substrate (concentration) [3H] SAM (35 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: [3H] (IC50: 22 μM) G9a (h) [Yost et al. 2011] Source human recombinant (E. coli) Substrate histone H3 full length (5 nM) Co-substrate [3H] SAM (25 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: SAH (2.1 μM) hSMYD2 [An et al. 2011] Source human recombinant (Sf9 cells) Substrate histone H4 full length (25 nM) Co-substrate [3H] SAM 150 nM Incubation 10 min/RT Detection method Scintillation counting Reference: SAH (0.16 μM) MLL complex (h) [Jiang et al. Source human recombinant (E. coli) 2013] Substrate Histone H3 full length (35 nM) Tracer [3H] SAM (200 nM) Incubation 30 min/RT Detection method Scintillation counting Reference: [3H] (0.97 μM) MLL2(h) complex [Jiang et al. Source human recombinant (E. coli) 2013] Substrate Histone H3 full length (35 nM) Tracer [3H] SAM (200 nM) Incubation 30 min/RT Detection method Scintillation counting Reference: SAH (0.97 μM) MLL3(h) complex [Ali et al. Source human recombinant (E. coli) 2014] Substrate Core histone (20 nM) Tracer [3H] SAM (200 nM) Incubation 20 min/RT Detection method Scintillation counting Reference: SAH (0.97 μM) MLL4(h) complex [Ali et al. Source human recombinant (E. coli) 2014] Substrate Core histone (30 nM) Tracer [3H] SAM (200 nM) Incubation 10 min/RT Detection method Scintillation counting Reference: SAH (0.97 μM) NSD1 (h) [Allali-Hassani et Source human recombinant (E. coli) al. 2014] Substrate Histone H3 full length (35 nM) Tracer [3H] SAM (200 nM) Incubation 30 min/RT Detection method Scintillation counting Reference: SAH (0.97 μM) NSD3/WHSC1L1 (h) [Selvi Source human recombinant (E. coli) et al. 2010] Substrate Histone H3 full length (35 nM) Tracer [3H] SAM (200 nM) Incubation 30 min/RT Detection method Scintillation counting Reference: [3H] (0.97 μM) PRDM9 (h) [Munoz-Fuentes Source human recombinant (E. coli) et al. 2011] Substrate Histone H3 full length (250 nM) Co-Substrate [3H] SAM (60 nM) Incubation 30 min/RT Detection method Scintillation counting Reference: SAH (370 μM) PRMT1 (h) [Cheng et al. Source human recombinant (E. coli) 2004] Substrate histone H4 full length (25 nM) Co-substrate [3H] SAM (60 nM) Incubation 90 min/RT Detection method Scintillation counting Reference: SAH (0.094 μM) PRMT3 (h) [Li et al. 2012] Source human recombinant (E. coli) Substrate histone H4 full length (25 nM) Co-substrate [3H] SAM (60 nM) Incubation 90 min/RT Detection method Scintillation counting Reference: SAH (0.86 μM) PRMT4 (h) [Selvi et al. 2010] Source human recombinant (Sf9 cells) Substrate histone H4 full length (25 nM) Co-substrate [3H] SAM (60 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: SAH (0.094 μM) PRMT5 complex (h) [Yost et al. Source human recombinant (Sf9 cells) 2011] Substrate histone H4 full length (250 nM) Co-substrate [3H] SAM (600 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: SAH (0.65 μM) PRMT6 (h) [Iberg et Source human recombinant (Sf9 cells) al. 2007] Substrate histone H4 full length (250 nM) Co-substrate [3H] SAM (600 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: SAH (0.65 μM) PRMT7 (h)[Zurita-Lopez Source human recombinant (Sf9 cells) et al. 2012] Substrate histone H2B (21-41) biotin labeled (4 nM) Co-substrate [3H] SAM (250 nM) Incubation 90 min/RT Detection method Scintillation counting Reference: SAH (0.65 μM) PRMT8 (h) [Lee et al. 2005] Source human recombinant (Sf9 cells) Substrate histone H4 full length (250 nM) Co-substrate [3H] SAM (600 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: SAH (0.65 μM) SETD2(h) [Du et al. 2008] Source human recombinant (E. coli) Substrate Nucleosome (0.5 μg/mL) Co-substrate [3H] SAM (250 nM) Incubation 10 min/RT Detection method Scintillation counting Reference: SAH (1.2 μM) SETD7 (h) [Li et al. 2012] Source human recombinant (E. coli) Substrate histone H3 full length (120 nM) Co-substrate [3H] SAM (850 nM) Incubation 30 min/RT Detection method Scintillation counting Reference: SAH (24 μM) SETD8 (h) [Yost et al. 2011] Source human recombinant (E. coli) Substrate histone H3 full length (120 nM) Co-substrate [3H] SAM (850 nM) Incubation 30 min/RT Detection method Scintillation counting Reference: SAH (24 μM) SETDB1 (h) [Schultz et al. Source human recombinant (Sf9 cells) 2002] Substrate histone H3 full length (15 nM) Co-substrate [3H] SAM (25 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: SAH (1.8 μM) SUV39H2 (h) [Yost et al. Source human recombinant (Sf9 cells) 2011] Substrate histone H3 full length (500 nM) Co-substrate [3H] SAM (350 nM) Incubation 120 min/RT Detection method Scintillation counting Reference: SAH (24 μM) SUV4-20H2 (h) [Li et al. Source human recombinant (E. coli) 2012] Substrate Nucleosome (1.5 μg/ml) Co-substrate [3H] SAM (75 nM) Incubation 15 min/RT Detection method Scintillation counting Reference: SAH (24 μM) WHSC1(h) (NSD2(h)) Source huma recombinant (E. coli) [Kang et al. 2009] Substrate Core Histone (1500 nM) Tracer [3H] SAM (250 nM) Incubation 15 min/RT Detection method Scintillation counting Reference: Chaetocin (IC50: 0.48 μM) Kinases Aurora B (h) (substrate Source Baculovirus histone H3 full length) Substrate Histone H3 full length (150 nM) [Sabbattini et al. 2007] Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (0.038 μM) DAPK3/ZIP (substrate Source Baculovirus histone H3 full length) Substrate Histone H3 full length(50 nM) [Preuss et al. 2003] Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.0073 μM) Haspin (h) (substrate histone Source human recombinant (E. coli) H3 full length) [Han et al. Substrate baculovirus Histone H3 full length (250 nM) 2011] Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.0073 μM) IKK alpha(h) (substrate Source human recombinant (Sf21 cells) histone H3 full length) [Baek 2011] Substrate Histone H3 full length (50 nM) Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.03 μM) MSK1(h)(substrate histone Source human recombinant insect cells H3 full length) [Baek 2011] Substrate Histone H3 full length (100 nM) Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.015 μM) MSK2(h) (substrate histone Source human recombinant (insect cells) H3 full length) [Baek 2011] Substrate Histone H3 full length (20 nM) Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.0058 μM) PIM1(h) (histone H3 full Source human recombinant (Sf21 cells) length substrate) [Baek 2011] Substrate Histone H3 full length (50 nM) Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.03 μM) PKBalpha/AKT1(h) Source human recombinant (Sf21 cells) (substrate histone H3 full length) Substrate Histone H3 full length (50 nM) [Barnett et al. 2005] Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.03 μM) PKBbeta/AKT2(h) Source human recombinant (baculovirus) (substrate histone H3 full Substrate Histone H3 full length (100 nM) length) [Barnett et al. 2005] Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.03 μM) PKBganuna/AKT3(h) Source human recombinant (baculovirus) (substrate histone H3 full Substrate Histone H3 full length (150 nM) length) [Baek 2011] Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.03 μM) Rsk2(h) (substrate histone H3 Source human recombinant (baculovirus) full length) [Baek 2011] Substrate Histone H3 full length (250 nM) Tracer [33P] ATP Incubation 10 min/RT Detection method Scintillation counting Reference: Staurosporine (IC50: 0.007 μM) Small molecule methyltransferases Catechol O-methyltransferase Source human recombinant (E. coli) (h) Substrate Pyrocatechol (250 nM) Tracer SAM (10 μM) Incubation 15 min/37° C. Detection method MS Reference: SAH-d4 (IC50: 14 μM) Glycine N- Source human recombinant (E. coli) methyltransferase (h) Substrate Glycine (100 μM) Tracer SAM (20 μM) Incubation 30 min/22° C. Detection method MS Reference: SAH-d4 (IC50: 17 μM) Guanidinoacetate N- Source human recombinant (E. coli) methyltransferase (h) Substrate Guanidineacetic acid (4 μM) Tracer SAM (7 μM) Incubation 30 min/22° C. Detection method MS Reference: SAH-d4 (IC50: 2.3 μM) Histamine N- Source human recombinant (E. coli) methyltransferase (h) Substrate Histamine (4 μM) Tracer SAM (4 μM) Incubation 30 min/22° C. Detection method MS Reference: SAH-d4 (IC50: 13 μM) Nicotinamide N- Source human recombinant (E. coli) methyltransferase (h) Substrate Nicotinamide (8 μM) Tracer SAM (6 μM) Incubation 15 min/22° C. Detection method MS Reference: SAH-d4 (IC50: 13 μM) Phenylethanolamine N- Source human recombinant (E. coli) methyltransferase (h) Substrate DL-Normetanephrine SAM (35 μM) Tracer SAM (6 μM) Incubation 45 min/22° C. Detection method MS Reference: SAH-d4 (IC50: 13 μM) Thiopurine S- Source human recombinant (E. coli) methyltransferase (h) Substrate 6-mercaptopurine (1.5 μM) Tracer SAM (1.5 μM) Incubation 30 min/22° C. Detection method MS Reference: SAH-d4 (IC50: 2.3 μM) Ubiquitin modifying enzymes BAP1 (h) [Misaghi et al. Source human recombinant (E. coli) 2009] Substrate LanthaScreen DUB substrate Substrate concentration 20 nM Incubation 180 min/RT Detection method LanthaScreen Reference: Ubiquitin aldehyde (IC50: 0.23 μM) USP 10 (h) [Horton et al. Source human recombinant (Sf9 cells) 2007] Substrate LanthaScreen DUB substrate Substrate concentration 10 nM Incubation 60 min/22° C. Detection method LanthaScreen Reference: Ubiquitin aldehyde (IC50: 0.23 μM) USP 14 (h) [Hu et al. 2005] Source human recombinant (Sf9 cells) Substrate LanthaScreen DUB substrate Substrate concentration 10 nM Incubation 240 min/RT Detection method LanthaScreen Reference: Ubiquitin Aldehyde (0.004 μM) USP 16 (h) [Horton et al. Source Human recombinant HEK 293 cells 2007] Substrate LanthaScreen DUB substrate Substrate concentration 20 nM Incubation 180 min/22° C. Detection method LanthaScreen Reference: Iodoacetamide (3.5 μM) USP 21(h) [Ye et al. 2011] Source human recombinant (E. coli) Substrate LanthaScreen DUB substrate Substrate concentration 10 nM Incubation 15 min/RT Detection method LanthaScreen Reference: Ubiquitin aldehyde (IC50: 0.17 μM) USP 7 (h) [Tian et al. 2011] Source human recombinant (Sf21 cells) Substrate LanthaScreen DUB substrate Substrate concentration 10 nM Incubation 60 min/RT Detection method LanthaScreen Reference: Ubiquitin Aldehyde (0.11 μM)

Results for the binding assays are shown below in Table 4.

TABLE 4 Inhibition of Compounds 1 to 3 at 10 μM on binding of epigenetic interacting protein modules against reference compounds. % Inhibition^(A) Reference Comp Comp Comp IC₅₀ Binding Assay 1 2 3 Compound μM Bromodomain ATAD2B (h) 52 25 28 Ischemin sodium 76.6 salt ASH1L (h) 95 59 42 JQ-1 13.8 BAZ2A (h) 56 17 16 GSK2801 1.8 BRPF1-1 (h) −25 −25 −4 Bromosporine 0.026 CECR2 (h) 100 25 15 Bromosporine 0.062 EP300 (h) 89 37 40 SGC-CBP30 0.050 KAT2A 76 19 18 JQ-1 45 (GCN5L2) (h) PCAF (h) 37 16 3 JQ-1 12 PB1(2) (h) 31 22 13 PFI-3 68 PB1(3) (h) 38 13 6 JQ-1 47 PB1(4) (h) 41 −3 16 Ischemin sodium 6.8 salt PHIP(2) (h) 61 30 23 SGC-CBP30 0.130 SP140 (h) 84 43 1 Ischemin 86 SMARCA2 (h) 83 21 18 PFI-3 0.47 TAF1(1) (h) 48 17 17 GSK2801 4.4 SMARCA4 (h) 53 16 13 PFI-3 3 TAF1(2)(h) 62 28 22 GSK2801 10 BAZ2B (h) 58 23 21 GSK2801 1.8 ATAD2A (h) 100 16 9 JQ-1 70 BRD2(1) (h) 60 89 19 JQ-1 0.510 BRD2(2) (h) 58 22 18 JQ-1 0.017 BRD3(1) (h) 47 17 13 JQ-1 0.040 BRD3(2) (h) 35 25 1 PFI-1 0.79 BRD4(1) (h) 49 11 8 JQ-1 0.018 BRD4(2) (h) 76 16 13 PFI-1 1.3 BRDT(1) (h) 43 23 29 JQ-1 0.110 CREBBP (h) 65 21 17 SGC-CBP30 0.120 FALZ (h) 42 18 5 I-CBP112 24.5 Chromodomain CBX1 (h) 15 0 −1 NA CBX2 (h) 17 6 3 NA CBX4 (h) 25 29 40 NA CBX6 (h) −1 27 58 NA CBX5 (h) 25 24 25 NA CBX7 (h) 25 33 43 NA CBX8 (h) 27 31 23 NA MBT domain L3MBTL1 41 61 59 NA L3MBTL3 (h) 22 29 33 NA PHD domain SP140 (h) 74 0 2 NA TRIM 33 (h) 59 3 −2 NA UHRF1 73 34 29 NA (108-286) (h) Tudor domain PHF20(1) 7 2 2 NA ^(A)% Inhibition of Control Specific Binding NA Not-applicable

Representative inhibition data for Compound 1 on binding example epigenetic targets is shown in Tables 5 to 8.

TABLE 5 ASH1L (h) bromodomain Conc. 1^(st) 2^(nd) Mean 3.0E−09M −2.6 −8.2 −5.4 3.0E−08M −12.3 1.8 −5.2 3.0E−07M −0.2 −2.9 −1.6 3.0E−06M 17.0 28.0 22.5 3.0E−05M 100.6 99.6 100.1

TABLE 6 CECR2(h) bromodomain binding curve Conc. 1^(st) 2^(nd) Mean 3.0E−09M 5.9 2.4 4.2 3.0E−08M −0.9 −1.7 −1.3 3.0E−07M −1.5 0.9 −0.3 3.0E−06M 7.3 8.6 7.9 3.0E−05M 99.4 99.6 99.5

TABLE 7 SP140 (h) bromodomain binding curve. Conc. 1^(st) 2^(nd) Mean 3.0E−09M −6.0 −8.6 −7.3 3.0E−08M −9.7 −14.6 −12.1 3.0E−07M −14.1 −11.0 −12.6 3.0E−06M 8.4 11.3 9.9 3.0E−05M 102.3 102.3 102.3

TABLE 8 UHRF1(108-286) (h) binding curve. Conc. 1^(st) 2^(nd) Mean 3.0E−09M −11.9 −13.1 −12.5 3.0E−08M −13.3 −12.6 −12.9 3.0E−07M −12.1 −13.2 −12.7 3.0E−06M −1.8 −0.1 −0.9 3.0E−05M 81.5 83.3 82.4

TABLE 9 IC₅₀ values from inhibition/concentration-response curves of compounds 1 and 2 for binding of epigenetic interacting protein modules against reference compounds. Compound 1 2 Reference IC₅₀ IC₅₀ Reference IC₅₀ (μm) (μm) Comp. (μm) Bromodomain ASH1L (h) 4.2 JQ-1 8.7 ATAD2A (h) 3.1 Ischemin 5.2 BRD2(1) (h) 22 1-BET 151 0.028 BRD4(2) (h) 5.6 PFI-1 1.1 CECR2(h) 5 Bromosporine 0.14 CREBBP (h) 3.4 SGC-CBP30 0.046 EP300 (h) 4.5 SGC-CBP30 0.068 KAT2A (GCN5L2) 3.9 GSK2801 10 (h) SP140 (h) 3.8 Ischemin 11 PHD domain SP140 (h) 4.6 NA NA UHRF1(108-286) (h) 11 NA NA

TABLE 10 Inhibition of compounds 1 to 3 at 10 μM on activity of epigenetic enzymes against references. % Inhibition^(A) Reference Comp Comp Comp IC₅₀ Assay 1 2 3 Compound μm Cell-based detection methyl modifications H3K27 ac −16 −12 −11 (Not-applicable) NA H3K27 me2-1 −2 3 2 NA H3K27 me3 −8 −7 0 NA H3K36 me2 10 7 −8 NA H3K4 me2 −5 −1 −9 NA H3K79 me2 −2 −16 −5 NA H3K9 ac 17 3 −1 NA H3K9 me2 1 11 20 NA Demethylases (KDMs) FBXL10 (h) 37 7 22 2,4 PDCA 0.61 FBXL11 (h) 34 2 11 2,4 PDCA 1.5 JARID1A (h) 64 −9 3 2,4 PDCA 0.18 JARID1B (h) 70 6 −2 2,4 PDCA 0.15 JARID1C (h) 49 −13 25 2,4 PDCA 0.23 JMJD1A (h) 80 37 −1 2,4 PDCA 0.76 JMJD2A (h) 29 −10 12 2,4 PDCA 0.083 JMJD2B (h) 65 16 8 2,4 PDCA 0.14 JMJD2C (h) 35 2 8 2,4 PDCA 0.091 JMJD2D (h) 36 −17 18 2,4 PDCA 0.091 JMJD2E (h) 65 10 6 2,4 PDCA 0.061 JMJD3 (h) 91 9 11 2,4 PDCA 52 LSD1 (h) 10 10 −3 Tranylcypromine 22 PHF8(h) 36 −11 13 Daminozide 0.28 UTX (h) 68 5 27 IOX1 0.15 DNA methyltransferases (DNMTs) DNMT1 (h) 10 4 −5 SAH 0.23 DNMT3b −3 −17 −4 SAH 0.094 DNMT3B/DNMT3L (h) 23 27 −20 SAH 0.045 hDNMT3a 3 3 −31 SAH 0.052 Histone acetyltransferases (HATs) CREBBP(h) 74 78 43 Garcinol 1.5 GCN5L2(h) 11 5 1 Anacardic Acid 4.7 HAT1(h) −3 3 −3 Garcinol 1.5 MYST3(h) 18 11 3 Garcinol 1.8 MYST4(h) 25 39 71 Curcumin 11 pCAF(h) −25 −26 −20 Garcinol 2.9 TIP60(h) 20 10 −3 Garcinol 1.5 Histone deacetylase (HDACs) HDAC1 (h) −2 −1 −1 trichostatin A 0.0052 HDAC2 (h) 1 0 −2 trichostatin A 0.024 HDAC3 (h) 1 1 3 trichostatin A 0.0068 HDAC4 (h) −73 −20 −11 trichostatin A 4.0 HDAC5 (h) −3 −2 −1 trichostatin A 1.0 HDAC6 (h) 12 2 −18 trichostatin A 0.0074 HDAC7 (h) −111 −6 16 trichostatin A 1.8 HDAC8 (h) −8 −19 0 trichostatin A 0.41 HDAC9 (h) −50 −1 −9 trichostatin A 9.1 HDAC10 (h) 1 3 6 trichostatin A 0.0090 HDAC11 (h) 27 −47 −130 scriptaid 8.9 Sirtuins sirtuin 1 (h) 2 −3 −5 suramin 7.9 (inhibitor effect) sirtuin 2 (h) −4 6 −19 suramin 21 (inhibitor effect) sirtuin 3 (h) −14 −8 −3 niacinamide 21 (inhibitor effect) Sirtuin 6 (h) 2 6 −4 EX-527 550 Sirtuin 7 (h) 7 −2 −8 JFD00244 1300 Methyltransferases (MTs) ASH1L 3 20 −54 SAH 9.1 DOT1L (h) −5 18 13 SAH 0.14 EHMT1 (h) 13 9 63 SAH 0.18 EZH1/EED/SUZ12 (h) −1 3 −12 SAH 8.4 EZH2/EED/SUZ12 (h) 19 26 4 SAH 22 (PRC2 complex) G9a (h) 86 64 49 SAH 2.1 hSMYD2 12 5 −12 SAH 0.16 MLL complex (h) 2 22 11 SAH 0.97 MLL2(h) complex 6 3 −15 SAH 24 MLL3(h) complex −22 −21 −33 SAH 9.0 MLL4(h) complex −16 −18 −4 SAH 0.55 NSD1 (h) 69 29 −10 chaetocin 0.13 NSD3/WHSC1L1 (h) −17 11 −35 Suramin 1.5 PRDM9 (h) 9 5 4 SAH 370 PRMT1 (h) 16 26 −1 SAH 0.094 PRMT3 (h) 22 50 14 SAH 0.86 PRMT4 (h) 90 84 77 SAH 0.094 PRMT5 complex (h) 8 −8 9 SAH 0.65 PRMT6 (h) 49 57 34 SAH 0.053 PRMT7 (h) −2 0 −36 SAH 0.86 PRMT8 (h) 5 24 −26 SAH 0.14 SETD2(h) −9 −4 −11 SAH 1.2 SETD7 (h) 12 16 −2 SAH 24 SETD8 (h) 4 25 −10 mercurochrome 2.4 SETDB1 (h) 53 43 52 SAH 1.8 SUV39H2 (h) −8 −16 14 SAH 24 SUV4-20H2 (h) 11 20 −45 SAH 4.7 WHSC1(h) (NSD2(h)) 3 14 7 Chaetocin 0.48 Kinases Aurora B (h) (substrate 6 4 −1 Staurosporine 0.038 histone H3 full length) DAPK3/ZIP (substrate 4 9 8 Staurosporine 0.0073 histone H3 full length) Haspin (h) (substrate 2 11 7 Staurosporine 0.032 histone H3 full length) IKK alpha(h) (substrate 26 28 14 Staurosporine 0.03 histone H3 full length) MSK1(h) (substrate 6 2 10 Staurosporine 0.015 histone H3 full length) MSK2(h) (substrate 9 12 −1 Staurosporine 0.0058 histone H3 full length) PIM1(h) (histone H3 22 11 5 Staurosporine 0.025 full length substrate) PKBalpha/AKT1(h) −1 −19 −15 Staurosporine 0.0073 (substrate histone H3 full length) PKBbeta/AKT2(h) 13 11 12 Staurosporine 0.021 (substrate histone H3 full length) PKBgamma/AKT3(h) 5 −7 3 Staurosporine 0.03 (substrate histone H3 full length) Rsk2(h) (substrate 5 7 −4 Staurosporine 0.007 histone H3 full length) Small molecule methyltransferases Catechol O- 6 1 0 SAH-d4 14 methyltransferase (h) Glycine N- 55 5 −11 SAH-d4 17 methyltransferase (h) Guanidinoacetate N- 8 −1 −1 SAH-d4 2.3 methyltransferase (h) Histamine N- 18 −2 18 SAH-d4 13 methyltransferase (h) Nicotinamide N- 13 −2 −2 SAH-d4 13 methyltransferase (h) Phenylethanolamine N- 23 9 5 SAH-d4 3.9 methyltransferase (h) Thiopurine S- 40 28 19 SAH-d4 2.3 methyltransferase (h) Ubiquitin modifying enzymes BAP1 (h) 1 −9 24 Ubiquitin 0.23 Aldehyde USP 10 (h) 7 2 −7 N- 1000 Methylmaleimide USP 14 (h) 1 0 −24 Ubiquitin 0.004 Aldehyde USP 16 (h) 11 14 12 Iodoacetamide 3.5 USP 21 (h) −18 3 3 Ubiquitin 0.17 aldehyde USP 7 (h) −18 −14 9 Ubiquitin 0.11 Aldehyde ^(A) % Inhibition of Control Values

TABLE 11 IC₅₀ values from inhibition/concentration-response curves of compounds 1, 2 and 4 for inhibition of epigenetic enzymes against reference compounds. Compound 1 2 4 Reference Enzyme and Cell-based IC₅₀ IC₅₀ IC₅₀ Reference IC₅₀ assays (μM) (μM) (μM) Compound (μM) Demethylases JARID1A (h) 9.6 2,4 PDCA 0.099 JARID1B (h) 3.6 2,4 PDCA 0.12 JMJD1A (h) 4.5 2,4 PDCA 3.9 JMJD2B (h) 12 2,4 PDCA 0.095 JMJD2E (h) 4.7 2,4 PDCA 0.11 JMJD3 (h) 14 2,4 PDCA 73 UTX (h) 6.7 IOX1 0.056 Histone acetyltransferases CREBBP(h) 3.9 6.1 Garcinol 5.4 MYST4 (h) 4.2 Curcumin 8.9 Histone methyltransferases G9a (h) 11 5.4 SAH 4.6 NSD1 (h) 4.9 chaetocin 0.24 PRMT4 (h) 4.7 5.4 4.7 SAH 0.056 Results: Epigenetic Targets, Diseases and Cancer

Epigenetic modifications can have a role in the development of a variety of diseases. Epigenetic regulation involves hierarchical covalent modification of DNA and proteins that package such as histones. DNA is regulated by methylation and demethylation on the cytosine residues. DNA methylation is mediated by members of the DNA-methyl transferase family (DNMT1, DNMT3A and DNMT3B) whereas demethylation is mediated by the family ten-eleven translocation (TET1-3). Besides methylation and demethylation, histone is also regulated by acetylation and deacetylation. The key proteins that mediate epigenetic signaling though acetylation and methylation of histones comprise histone acetyltransferases (HATs), histone deacetylases (HDMs), protein methyltransferases (PMTs) including lysine methyltransferases (KMTs) protein arginine methyltransferases (PRMTs), and bromodomain-containing proteins and proteins that bind to methylated histones (Arrowsmith et al., 2012; Plass et al., 2013; Tough et al., 2014; Biggar and Li, 2015).

Bromodomain Containing Proteins

Compound 1 showed significant inhibitory activity towards many of the bromodomain containing proteins (Table 10), in particular ASH1L, CECR2, EP300, KAT2A, PHIP(2), SP140, SMARCA2, TAF1(2), ATAD2A, BRD2(1), BRD4(2) and CREBBP. Compound 2 only showed significant activity towards BRD2(1). Recent studies have implicated bromodomain (BRD) containing proteins in a wide range of human diseases, including cancer (Taverna et al., 2007; Prinjha et al., 2012; Biggar and Li, 2015). The most investigated member of BRD containing proteins (BCPs) as drug targets is the BET family proteins. Currently, there are several BET inhibitors in various stages of clinical trials including RVX-208, I-BET 762, OTX 015, CPI-0610 and TEN-010 (see Table 14 in Tough et al., 2014).

In addition, JQ1 and I-BET have been shown to interact with NF-κB and induce apoptosis in drug resistant leukemia (Ciceri et al., 2014). NF-κB plays a central role inflammation and unresolved inflammation is involved in many disease states including cancer.

Several bromodomain containing proteins, including ASH1L, ATAD2A/B, BAZ2A/B, CECR2, EP300, KAT2A, PHIP(2), PB1, SMARCA2/4, BRD2/4 and CREBBP, have been shown to be up-regulated in many types of cancers (Tough et al., 2014; Fu et al, 2015). Hence, BRDs are therefore therapeutic targets for cancer. BRD-containing domains have been linked to the development of a number of extremely aggressive tumours containing BRD4-NUT and BRD3-NUT. CREBBP mutations have been identified in relapsed acute lymphoblastic leukaemia and are very common in diffuse large B-cell lymphoma and follicular lymphoma, Hodgkin's lymphoma. CREBBP and the related HAT are highly expressed in advanced prostate cancer and expression levels have been linked with patient survival. ATAD2 is over expressed in more than 70% of breast tumours and higher protein levels correlate with poor overall survival and disease recurrence (Ciro et al., 2009). ATAD2B is highly expressed in glioblastoma and oligodendroglioma as well as breast carcinoma (Krakstad et al., 2015). BRD4 is linked to development of cervical cancer (Weidner-Glunde et al.; 2010).

Compound 1 showed good inhibitory activity against domain PHD (plant homology domain) containing domain proteins SP140, TRIM 33, UHRF1 (108-286)).

Methyltransferases

Compound 1 was a strong inhibitor of the lysine methyltransferase enzyme G9a whereas compounds 2 and 3 were weaker (Table 10). G9a is a multipotent regulator of gene expression which has been shown to be over expressed in many different types of cancers (See reviews for details: Shankar et al., 2013; Casciello et al., 2015). Compounds 1 to 3 strongly inhibited the activity of PRMT4 and moderately inhibited the activity of PRMT6. Dysregulated PRMT expression and activity have been observed in a variety of cancers and PRMT1-5 and 7 have been shown to be overexpressed or otherwise contribute to tumorigenesis whereas PRMT8, and 9 have not been implicated in oncogenesis (Fuhrmann et al., 2015). PRMT4 (CARM1) is necessary for NFκB target gene expression. Further study showed a link between PRMT4 and p300 acetyltransferase activity in NFκB recruitment and gene activation.

Demethylases

Compound 1 showed significant inhibitory activity towards many of the lysine demethylase enzymes (Table 10), in particular JARID1A, JARID1B, JMJD1A, JMJD2B, JMJD2E, JMJD3 and UTX. Aberrant expression and mutations of lysine demethylases have been linked to various cancers (Hojfeldt et al., 2013; Tough et al., 2014). Mutation of lysine demethylases including FBXL10, JMJD2A, JMJDB, JMJD2C, JARID1B and PHF2 have been shown to be overexpressed in breast, colorectal, lung, prostrate, bladder and other tumours; the functional significance of JMJD2C overexpression is further suggested by the presence of the JMJD2C gene within an amplified region of a chromosome in multiple cancers (Xiang et al., 2007; Couvelard el al., 2008; Roesch et al., 2010; He et al., 2011a; Berry and Janknecht, 2013; Kogure et al., 2013; Tzatsos et al., 2013).

Histone Acetyltransferases

The serrulatanes diterpenes showed significant inhibitory activity on histone acetyltransferases CREBBP and MYST4 (Table 10). Misregulation of histone acetyltransferase activity has been linked to many different pathogenic states including cancers, neurodegenerative disorders, and metabolic, respiratory, inflammatory and cardiovascular diseases (Adcock and Lee, 2006; Avvakumov and Cote, 2007; Grabiec et al., 2008; Ghizzo et al., 2011, Iyer et al., 2011; Pirooznia and Elefant, 2013).

TABLE 12 Enzyme and Cell-based stimulation assays for compounds 1 to 3 at 10 μM concentration % Stimulation Relative Reference to Control EC₅₀ Assay Comp 1 Comp 2 Comp 3 Compound (μM) H3K9 ac (increase) −21 −26 −8 (Not-applicable) NA noneH3K27 ac −28 −17 −25 NA (increase) sirtuin 1 (h) (activator −5 −3 −4 Resveratrol 27 effect) Pharmacology Assays

The in vitro pharmacological studies were conducted by Eurofins PanLab (Taiwan) using the parameters shown in Table 13 with respect to appropriate literature references.

TABLE 13 Pharmacology assay methods. Angiotensin system Angiotensin AT2 [Lee et al. 2001] Source Human recombinant CHO-K1 cells Vehicle 1.00% DMSO Ligand 0.050 nM [¹²⁵I] CGP-42112A Non-Specific Ligand 10.0 μM (Sar¹, Ile⁸)-Angiotensin II Incubation 3 hours @ 37° C. (Time/Temperature) Incubation Buffer 50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 1 mM EDTA, 0.1% BSA Quantitation Method Radio ligand Binding Angiotensin, AT₁/ACE [Rubin et al. 1978] Source Dunkin Hartley Guinea pig 600 ± 80.0 g ileum Vehicle 0.10% DMSO Incubation 5 minutes @ 32° C. (Time/Temperature) Incubation Buffer Krebs, pH 7.4 Quantitation Method Isotonic (cm changes) Peptidase, Angiotensin Converting Enzyme [Bunning et al. 1983] Source Rabbit lung Substrate 500 μM (N-3[2-furyl] acryloyl)-Phe-Gly-Gly (FAPGG) Vehicle 1.00% DMSO Pre incubation 15 minutes @ 25° C. (Time/Temperature) Incubation 30 minutes @ 25° C. (Time/Temperature) Incubation Buffer 50 mM HEPES, pH 7.5, 300 mM NaCl Quantitation Method Spectrophotometric quantitation of FAPGG Chemokines Chemokine CCR1 [Hesselgesser et al. 1998] Source Human recombinant Chem-2 cells Vehicle 1.00% DMSO Ligand 0.10 nM [¹²⁵I] MIP-1α Non-Specific Ligand 0.10 μM MCP-3 Incubation 3 hours @ 25° C. (Time/Temperature) Incubation Buffer 50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA Quantitation Method Radio ligand Binding Chemokine CCR2B [Gong et al. 1997] Source Human recombinant CHO-K1 cells Vehicle 1.00% DMSO Ligand 0.10 nM [¹²⁵I] MCP-1 Non-Specific Ligand 0.030 μM MCP-1 Incubation 60 minutes @ 25° C. (Time/Temperature) Incubation Buffer 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA Quantitation Method Radio ligand Binding Chemokine CX3CR1 [Combadiere et al. 1998] Source Human recombinant Chem-1 cells Vehicle 1.00% DMSO Ligand 20.0 pM [¹²⁵I] Fractalkine Non-Specific Ligand 10 nM Fractalkine Incubation 90 minutes @ 25° C. (Time/Temperature) Incubation Buffer 50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA, 0.1% NaN3 Quantitation Method Radio ligand Binding Chemokine CXCR1/2 (IL-8, Non-Selective) [Grob et al. 1990] Source Human neutrophils Vehicle 1.00% DMSO Ligand 15.0 pM [¹²⁵I] IL-8 Non-Specific Ligand 10 nM IL-8 Incubation 2 hours @ 4° C. (Time/Temperature) Incubation Buffer 50 mM Tris-HCl, pH 7.4 1 mM EDTA, 5 mM MgCl2, 5 mg/ml BSA Quantitation Method Radio ligand Binding Chemokine CXCR2 (IL-8RB) [Ahuja and Murphy 1996] Source Human recombinant CHO-K1 cells Vehicle 1.00% DMSO Ligand 15.0 pM [¹²⁵I] IL-8 Non-Specific Ligand 10 nM IL-8 Incubation 60 minutes @ 25° C. (Time/Temperature) Incubation Buffer 25 mM HEPES, pH 7.4, 2 mM CaCl2, 1 mM MgCl2, 0.2% BSA Quantitation Method Radio ligand Binding Chemokine CXCR4 [Valenzuela-Fernandez 2002] Source Human recombinant Chem-1 cells Vehicle 1.00% DMSO Ligand 0.030 nM [¹²⁵I] SDF-1α Non-Specific Ligand 0.030 μM SDF-1α Incubation 90 minutes @ 25° C. (Time/Temperature) Incubation Buffer 50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA Quantitation Method Radio ligand Binding Histone Deacetylases (HDACs) Deacetylase, Histone 1 [Strahl and Allis 2000] Source Human recombinant Insect Sf9 cells Substrate 25.0 μM Fluor-de-Lys deacetylase Vehicle 1.00% DMSO Pre incubation 15 minutes @ 25° C. (Time/Temperature) Incubation 60 minutes @ 25° C. (Time/Temperature) Incubation Buffer 25 mM Tris-HCl, pH 8.0, 2.7 mM KCl, 1 mM MgCl2, 137 mM NaCl Quantitation Method Spectrofluorimetric quantitation of Fluor- de-Lys deacetylsubstrate Deacetylase, Histone 2 [Strahl and Allis 2000] Source Human recombinant Insect Sf9 cells Substrate 25.0 μM Fluor-de-Lys deacetylase Vehicle 1.00% DMSO Pre incubation 15 minutes @ 25° C. (Time/Temperature) Incubation 60 minutes @ 25° C. (Time/Temperature) Incubation Buffer 25 mM Tris-HCl, pH 8.0, 2.7 mM KCl, 1 mM MgCl2, 137 mM NaCl Quantitation Method Spectrofluorimetric quantitation of Fluor- de-Lys deacetylsubstrate Growth Factor Epidermal Growth Factor (EGF) [Dittadi et al. 1990] Source Human A431 cells Vehicle 1.00% DMSO Ligand 0.080 nM [¹²⁵I] EGF (human) Non-Specific Ligand 0.10 μM EGF (human) Incubation 60 minutes @ 25° C. (Time/Temperature) Incubation Buffer 50 mM HEPES, pH 7.7, 0.1% BSA, 1.2 mM CaCl2, 5 mM KCl, 1.2 mM MgSO4, 138 mM NaCl Quantitation Method Radio ligand Binding Other classes Catechol-O-Methyl Transferase (COMT) [Muller-Enoch et al. 1976] Source Porcine liver Substrate 1.0 μM Esculetin Vehicle 1.00% DMSO Pre incubation 15 minutes @ 37° C. (Time/Temperature) Incubation 30 minutes @ 37° C. (Time/Temperature) Incubation Buffer 36 mM Tris-HCl, pH 8.0, 1.35 mM DTT, 0.54 mM MgCl2, 30.6 μM SAM Quantitation Method Spectrofluorimetric quantitation of scopoletin Phospholipase sPLA2-V [Tietge et al. 2005] Source Human E. coli Substrate 250 μM diheptanoyl thio-PC Vehicle 1.00% DMSO Pre incubation 15 minutes @ 25° C. (Time/Temperature) Incubation 30 minutes @ 25° C. (Time/Temperature) Incubation Buffer 16.25 mM Tris-HCl, 6.5 mM CaCl2, 65 mM KCl, 0.2 mM Triton X-100 Quantitation Method Spectrophotometric quantitation of heptanoyl thio-PC Protein Tyrosine Phosphatase, PTPN1 (PTP1B) [Montalibet et al. 2005] Source Human recombinant E. coli Substrate 10.0 μM DiFMUP Vehicle 1.00% DMSO Pre incubation 15 minutes @ 37° C. (Time/Temperature) Incubation 60 minutes @ 37° C. (Time/Temperature) Incubation Buffer 50 mM HEPES, pH 7.2, 0.1% BSA, 1 mM DTT Quantitation Method Spectrofluorimetric quantitation of DiFMU Steroid 5α-Reductase [Sun and Tu 1998] Source Wistar Rat liver Substrate 0.90 μM Testosterone Vehicle 1.00% DMSO Pre incubation 15 minutes @ 37° C. (Time/Temperature) Incubation 30 minutes @ 37° C. (Time/Temperature) Incubation Buffer 40 mM Potassium Phosphate, pH 6.5 containing 1 mM DTT, 50 μM NADPH Quantitation Method EIA quantitation of Testosterone Thioredoxin Reductase [Becker et al. 2000] Source E. coli Substrate 1.0 mM 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) Vehicle 1.00% DMSO Pre incubation 15 minutes @ 25° C. (Time/Temperature) Incubation 60 minutes @ 25° C. (Time/Temperature) Incubation Buffer 100 mM Potassium Phosphate, pH 7.4, 2 mM EDTA, 0.2 mg/ml BSA Quantitation Method Spectrophotometric quantitation of 5-Mercapto-2-nitrobenzoic acid Xanthine Oxidase [Hatano et al. 1990] Source Bovine buttermilk Substrate 165 μM Xanthine Vehicle 1.00% DMSO Pre incubation 15 minutes @ 25° C. (Time/Temperature) Incubation 30 minutes @ 25° C. (Time/Temperature) Incubation Buffer 33.3 mM Potassium Phosphate, pH 7.5 Quantitation Method Spectrophotometric quantitation of Uric acid Histamine H1 [De Backer et al., 1993] Source Human recombinant Chem-1 cells Substrate 1.00% DMSO Vehicle 1.20 nM [³H] Pyrilamine Pre incubation 1.0 μM Pyrilamine (Time/Temperature) Incubation 3 hours @ 25° C. (Time/Temperature) Incubation Buffer 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2 Quantitation Method Radio ligand Binding Histamine H2 [Ruat et al., 1990] Source Human recombinant CHO-K1 cells Vehicle 1.00% DMSO Ligand 0.10 nM [¹²⁵I] Aminopotentidine Non-Specific Ligand 3.0 μM Tiotidine Incubation 2 hours @ 25° C. (Time/Temperature) Incubation Buffer 50 mM Phosphate, pH 7.4 Quantitation Method Radio ligand Binding Histamine H3 [Krueger et al., 2005] Source Human recombinant Chem-1 cells Substrate 1.00% DMSO Vehicle 0.40 nM [³H] N-α-Methylhistamine (NAMH) Pre incubation 1.0 μM R(−)-α-Methylhistamine (RAMH) (Time/Temperature) Incubation 2 hours @ 25° C. (Time/Temperature) Incubation Buffer 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.1% BSA Quantitation Method Radio ligand Binding Histamine H4 [Liu et al., 2001] Source Human recombinant Chem-1 cells Substrate 1.00% DMSO Vehicle 8.20 nM [³H] Histamine Pre incubation 1.0 μM Histamine (Time/Temperature) Incubation 90 minutes @ 25° C. (Time/Temperature) Incubation Buffer 50 mM Tris-HCl, pH 7.4, 1.25 mM EDTA Quantitation Method Radio ligand Binding HMG-CoA Reductase [Kubo and Strott, 1987] Source Human recombinant E. coli Substrate 2.50 μM [¹⁴C]HMG-CoA Vehicle 1.00% DMSO Pre incubation 15 minutes @ 37° C. (Time/Temperature) Incubation 20 minutes @ 37° C. (Time/Temperature) Incubation Buffer 100 mM KH2PO4, pH 7.5, 8 mM G-6-P, 1 mM NADP, 4 mM EDTA, 2 mM DTT, 0.6 U/ml G-6-P-DH Quantitation Method Quantitation of [¹⁴C] Mevalonate 5-Lipoxygenase [Pufahl et al., 2007] Source Human recombinant Insect Sf9 cells Substrate 25.0 μM Arachidonic acid Vehicle 1.00% DMSO Pre incubation 5 minutes @ 25° C. (Time/Temperature) Incubation 20 minutes @ 25° C. (Time/Temperature) Incubation Buffer 50 mM Tris-HCl, pH 7.4, 5 mM CaCl2, 2 mM EDTA, 1 μM ATP Quantitation Method Spectrofluorimetric quantitation of rhodamine 123 Lipid Peroxidase [Mansuy et al., 1986] Source Dunkin Hartley Guinea pig liver microsomes Substrate Polyunsaturated fatty acid Vehicle 1.00% DMSO Pre incubation 15 minutes @ 37° C. (Time/Temperature) Incubation 20 minutes @ 37° C. (Time/Temperature) Incubation Buffer 0.25M Potassium Phosphate, pH 7.4, 0.1 mM EDTA Quantitation Method Spectrophotometric quantitation of Malondialdehyde Lipoxygenase 12-LO [Romano et al., 1993] Source Human platelets Substrate 30.0 μM Arachidonic acid Vehicle 1.00% DMSO Pre incubation 15 minutes @ 25° C. (Time/Temperature) Incubation 15 minutes @ 25° C. (Time/Temperature) Incubation Buffer 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100 Quantitation Method Spectrophotometric quantitation of 12-HETE Monoamine Oxidase MAO-A [Urban et al., 1991] Source Human recombinant Insect Hi5 cells Substrate 50.0 μM Kynuramine Vehicle 1.00% DMSO Pre incubation 15 minutes @ 37° C. (Time/Temperature) Incubation 60 minutes @ 37° C. (Time/Temperature) Incubation Buffer 100 mM Potassium Phosphate, pH 7.4 Quantitation Method Spectrofluorimetric quantitation of 4-hydroxyquinoline Monoamine Oxidase MAO-B [Urban et al., 1991] Source Human recombinant Insect Hi5 cells Substrate 50.0 μM Kynuramine Vehicle 1.00% DMSO Pre incubation 15 minutes @ 37° C. (Time/Temperature) Incubation 60 minutes @ 37° C. (Time/Temperature) Incubation Buffer 100 mM Potassium Phosphate, pH 7.4 Quantitation Method Spectrofluorimetric quantitation of 4-hydroxyquinoline Myeloperoxidase [Svensson et al., 1987] Source Human PMN leukocytes Substrate 20.0 mM Guaiacol Vehicle 1.00% DMSO Pre incubation 15 minutes @ 25° C. (Time/Temperature) Incubation 5 minutes @ 25° C. (Time/Temperature) Incubation Buffer 0.1M Sodium Phosphate, pH 7.4 Quantitation Method Spectrophotometric quantitation of Tetraguaiacol [3H]-2-Deoxy-D-glucose uptake[Yamamoto et al., 2006] Source Rat L6 skeletal muscle cells Substrate 0.10% DMSO Vehicle 24 hours @ 37° C. Pre incubation 15 minutes @ 37° C. (Time/Temperature) Incubation KRPH, pH 7.4 (Time/Temperature) Incubation Buffer Radiometric quantitation of insulin-induced 2-DG uptake Quantitation Method Rat L6 skelet al muscle cells Adhesion and Transcription response Adhesion, ICAM-1-Mediated [Cobb et al. 1992] Source Human Vehicle 0.40% DMSO Ligand 0.030 nM [¹²⁵I] SDF-1α Non-Specific Ligand 0.030 μM SDF-1α Incubation 30 minutes @ 37° C. (Time/Temperature) Incubation Buffer 25 mM HEPES, pH 7.4, RPMI-1640, 1% FBS Quantitation Method Spectrofluorimetric quantitation of adhesion Adhesion, VCAM-1-Mediated [Stoltenborg et al. 1994] Source Human Vehicle 0.40% DMSO Incubation 60 minutes @ 37° C. (Time/Temperature) Incubation Buffer 25 mM HEPES, pH 7.4, RPMI-1640, 1% FBS Quantitation Method Spectrofluorimetric quantitation of adhesion Transcription Response, NF-κB [Lenardo and Baltimore 1989] Source Human Vehicle 0.50% DMSO Incubation 4 hours @ 37° C. (Time/Temperature) Incubation Buffer RPMI-1640, pH 7.4 Quantitation Method Spectrofluorimetric quantitation of β- galactosidase

The results of pharmacological assays are shown in Table 14.

TABLE 14 Pharmacological activity of compounds 1 to 3 at 10 μM. % Control Response Assay Ascii Assay Name 1 2 3 Year Angiotensin system Angiotensin AT2 29 18 9 2015 Angiotensin, AT 1/ACE - Agonist 0 0 0 2015 Angiotensin, AT 1/ACE - Antagonist 34 10 17 2015 Peptidase, Angiotensin Converting 0 −3 −3 2015 Enzyme Chemokines Chemokine CCR1 33 −3 −5 2015 Chemokine CCR2B 1 −23 −10 2015 Chemokine CX3CR1 −1 5 7 2015 Chemokine CXCR1/2 (IL-8, Non- 30 −28 −1 2015 Selective) Chemokine CXCR2 (IL-8RB) 12 −7 1 2015 Chemokine CXCR4 14 −27 −13 2015 Histone Deacetylases (HDACs) Deacetylase, Histone 1 17 23 NA 2014 Deacetylase, Histone 1 −27 −18 −26 2015 Deacetylase, Histone 2 56 44 NA 2014 Deacetylase, Histone 2 34 21 12 2015 Deacetylase, Histone 3 −5 25 NA 2014 Deacetylase, Histone 4 4 −5 NA 2014 Deacetylase, Histone 5 −9 −11 NA 2014 Deacetylase, Histone 6 0 −17 NA 2014 Deacetylase, Histone 7 1 −16 NA 2014 Deacetylase, Histone 8 29 13 NA 2014 Deacetylase, Histone 9 −14 −13 NA 2014 Deacetylase, Histone 10 11 9 NA 2014 Deacetylase, Histone 11 9 5 NA 2014 Sirtuins Deacetylase, Sirtuin SIRT1 6 −3 NA 2014 Deacetylase, Sirtuin SIRT2 0 26 NA 2014 Deacetylase, Sirtuin SIRT3 −6 −9 NA 2014 Deacetylase, Sirtuin SIRT5 −16 24 NA 2014 Deacetylase, Sirtuin SIRT6 35 18 NA 2014 Growth Factor Epidermal Growth Factor (EGF) −8 −9 4 2015 Nitric Oxide Synthases Nitric Oxide Synthase, Endothelial 6 5 NA 2014 (eNOS) Nitric Oxide Synthase, Inducible (iNOS) 16 7 NA 2014 Nitric Oxide Synthase, Neuronal (nNOS) 14 5 NA 2014 Other classes Catechol-O-Methyl Transferase (COMT) 0 −1 3 2015 Histamine H1 85 66 NA 2014 Histamine H1 97 60 50 2015 Histamine H2 67 24 NA 2014 Histamine H2 73 −5 66 2015 Histamine H3 −10 −5 NA 2014 Histamine H3 12 24 1 2015 Histamine H4 50 16 NA 2014 Histamine H4 12 −1 10 2015 HMG-CoA Reductase 29 27 24 2015 5-Lipoxygenase 79 53 67 2015 Lipid Peroxidase 98 53 74 2015 Lipoxygenase 12-LO 60 40 29 2015 Monoamine Oxidase MAO-A 91 34 27 2015 Monoamine Oxidase MAO-B 75 39 18 2015 Myeloperoxidase 52 5 NA 2014 Myeloperoxidase 27 1 −7 2015 Phospholipase sPLA2-V 4 3 0 2015 Protein Tyrosine Phosphatase, 20 3 −3 2015 PTPN1 (PTP1B) Steroid 5alpha-Reductase 2 −6 3 2015 Thioredoxin Reductase 71 6 NA 2014 Thioredoxin Reductase 35 12 9 2015 Xanthine Oxidase 14 20 27 2015 Pharmacological Assays: Further Lipoxygenase, Lipid Peroxidase and Monoamine Oxidase Assays

Further in vitro pharmacological studies were conducted by Eurofins Panlabs (Taiwan) Ltd. using the parameters shown in Table 3 with respect to appropriate literature references.

For the Compound 1 5-lipoxygenase, lipid peroxidase and monoamine oxidase MAO-A assays, the inhibition/concentration-response curve concentrations were 10, 3, 1, 0.3, 0.1, 0.03 and 0.01 μM (Table 15).

TABLE 15 Activity of Compound 1 against 5-lipoxygenase, lipid peroxidase and monoamine oxidase (MAO-A) IC₅₀ (μM) and reference compounds Compound 1 Reference Assay IC₅₀ μM Compound IC₅₀ μM 5-Lipoxygenase 2.31 NDGA 0.64 Lipid Peroxidase 1.47 N-Propyl Gallate 206 Monoamine oxidase 6.74 Clorgyline 0.00143 A (MAO-A)

Compounds 1 to 3 are potent to moderately potent inhibitors of lipid oxidation (Tables 14 and 15). Therefore, they can be used in blocking inflammation induced by oxidative damage in cancer.

5-Lipoxygenase promotes lipid oxidation and produces leukotrienes. Compounds 1 to 3 are potent to moderately potent inhibitor of 5-lipoxygenase (Tables 14 and 15). Therefore, they can be used in blocking inflammation induced by oxidative damage and inflammatory pathophysiology in cancer.

12-Lipoxygenase promotes lipid oxidation and produces leukotrienes. Compounds 1 to 3 are moderately potent inhibitor of 12-lipoxygenase (Table 14). Therefore, they can be used in blocking inflammation induced by oxidative damage and inflammatory pathophysiology in cancer.

Compound 1 is a potent inhibitor of monoamine oxidase A and B (Tables 14 and 15). Therefore, it can be used in blocking oxidative damage induced by oxidation of monoamines and as an antidepressant in cancer.

Compounds 1 to 3 inhibit binding of the radioligand to histamine H1 and H2 receptors (Table 14). Therefore, they can be used in blocking inflammation induced by histamine resulting in anti-inflammatory action useful in cancer treatment.

Compound 1 inhibited the activity of thioredoxin reductase (Table 14). Inhibition of thioredoxin reductase activity can be used to selectively increasing oxidative stress in cancer cell while reducing oxidative stress in normal cells and

Compound 1 inhibited the activity of myeloperoxidase (Table 14). Inhibition of myeloperoxidase activity can be used to selectively reduce oxidative stress in immune cells during cancer therapy.

Curves showing binding against 5-lipoxygenase; lipid peroxidase; and monoamine oxidase (MAO-A) for Compound 1 are presented in FIGS. 7 to 9.

Pharmacology Assays: 2-Deoxy-D-Glucose Uptake into Muscle Cells

TABLE 16 Effect of compounds 1 to 4 at 10 μM on 2-deoxy-D-glucose uptake into muscle cells. % Control Response Assay Ascii Assay Name 1 2 3 4 Year [3H]-2-Deoxy-D-glucose −61 15 −52 −45 2016 uptake - agonist* [3H]-2-Deoxy-D-glucose 152 67 195 151 2016 uptake - antagonist* *agonist - Increase in 2-DG uptake relative to insulin response *antagonist - Inhibition of insulin-induced 2-DG uptake Pharmacology Assays: Adhesion and Transcription Response

TABLE 17 Dose response and IC₅₀ values for the pharmacological activity of representative serrulatane diterpenes. Compound 1 Compound 2 Compound 3 Conc Resp IC₅₀ Resp IC₅₀ Resp IC₅₀ Ascii Assay Name μM Av % μM Av % μM Av % μM Adhesion, ICAM-1- 10 6 >10 5 >10 7 >10 Mediated Adhesion, ICAM-1- 1 3 >10 4 >10 6 >10 Mediated Adhesion, ICAM-1- 0.1 −2 >10 3 >10 −4 >10 Mediated Adhesion, ICAM-1- 0.01 1 >10 0 >10 −5 >10 Mediated Adhesion, ICAM-1- 0.001 −4 >10 7 >10 −7 >10 Mediated Adhesion, VCAM-1- 10 −7 >10 −18 >10 −4 >10 Mediated - Antagonist Adhesion, VCAM-1- 1 −9 >10 −18 >10 7 >10 Mediated - Antagonist Adhesion, VCAM-1- 0.1 3 >10 −8 >10 13 >10 Mediated - Antagonist Adhesion, VCAM-1- 0.01 0 >10 −17 >10 18 >10 Mediated - Antagonist Adhesion, VCAM-1- 0.001 13 >10 −7 >10 13 >10 Mediated - Antagonist Transcription Response, 10 103 1.4 34 >10 −19 >10 NF-kappaB - Antagonist Transcription Response, 1 17 1.4 10 >10 1 >10 NF-kappaB - Antagonist Transcription Response, 0.1 0 1.4 −3 >10 −7 >10 NF-kappaB - Antagonist Transcription Response, 0.01 −5 1.4 −7 >10 −11 >10 NF-kappaB - Antagonist Transcription Response, 0.001 −25 1.4 −7 >10 −16 >10 NF-kappaB - Antagonist

Nuclear factor kappa B (NF-κB) was first discovered as a factor in the nucleus of B lymphocytes that binds to the enhancer of kappa light chain of immunoglobulin and is also a lymphoid specific (Sen and Baltimore, 1986). The NF-κB family includes RelA/p65, NF-κB1 p50/p105, NF-κB2 p52/p100, C-Rel and RelB. These proteins contain an N-terminal Rel homology domain (RFID) that is responsible for binding to DNA and other proteins and harbour a nuclear leading sequence (NLS). NF-κB proteins function as a dimeric transcription factor that regulates the expression of genes influencing a broad range of biological processes including innate and adaptive immunity, inflammation, stress responses, B-cell development and lymphoid organogenesis (Pantano et al., 2006; Brigelius-Fohê and Fohê 2011; Ghosh et al., 2012; Akdis et al., 2017).

NF-κB has been shown to have diverse and complex roles in cancer and immune modulation (Perkins, 2012; Sethi et al., 2012; Parri and Chiarugi, 2013; D'Ignazio el al., 2015). Based on significant inhibitory activity against NF-κB (IC₅₀ 0.69 μM) and cytokines (IC₅₀ 6.6, 8.8, 4.3, 8.9 μM, for IL1β, IL-6, IL10, TNFα respectively, Compound 1 can potentially influence the expression of the aforementioned biological processes as well as modulation of immune responses. In support for these results, Eurofins Oncopanel Genomic analysis showed Compound 1 clustered near to ABT-199, a BH3 domain inhibitor of BCL-2. ABT-199 blocked anti-apoptotic of BCL-2 leading to programmed cell death. In addition to antitumor effect of BCL-2 therapeutics, the BH3 mimetics have been proposed in the context of immune modulation (Ludwig et al., 2016). Taken together, Compound 1 could target cancer cell death as well as immune modulation. FIG. 10 shows transcription response and binding against NF-κB for Compound 1.

In Vitro Cell Viability Assay of Serrulatane Terpenes

Four serrulatane diterpenes, namely compounds 1 to 4, and Myoporum insulare resin were evaluated for in vitro inhibition of cell growth in 43 cancer cell lines and one normal cell line as shown in Table 19. The assay is a 72 hour (three day) assay.

Methodology of the In Vitro Cell Viability Assay

Cell viability assays were carried out by Eurofins PanLab (Taiwan). The assays are based on the established principle that cell viability (survival) can be evaluated by measuring the intracellular levels of adenosine triphosphate (ATP) by bioluminescence in metabolically active cells (Xia, M et al. 2008). Assays are carried out by seeding cells in two plates (To and T₇₂). At time “zero” cell plate (To) is harvested, treated, and incubated for 72 h (three days) when the T₇₂ plate will be harvested. The intracellular levels of ATP are measured and represent the amount of viable cells.

Cell viability assay results for compounds 1 to 4 and Myoporum insulare resin/exudate extract towards 43 cancer cell lines and 1 normal cell line are shown in Table 18.

TABLE 18 Inhibition of cell growth at 10.5 μg/mL of Myoporum insulare resin and 10 μM of compounds 1 to 4. % Cell growth Myoporum Cell lines insulare resin 1 2 3 4 Normal HUVEC, 32 −90 97 91 37 Endothelium Brain U-87 MG 36 7 101 111 81 SK-N-MC 4 −87 73 82 −92 Leukemia HL-60 (TB) 13 −62 −35 138 −83 K-562 −20 −55 69 93 60 MV-411 −96 −96 37 76 −94 MOLT-4 −97 −94 38 77 −93 Non-Small Cell Lung Cancer A549/ATCC 60 −92 102 106 93 LL/2 41 −62 105 111 98 NCI-H460 63 −37 88 100 93 PC-6 30 −62 92 96 4 Colon Cancer CT26.WT 36 2 120 103 33 SW-620 −94 −89 97 107 74 DLD-1 29 −65 95 101 68 HCT-15 15 −62 94 116 69 HCT-116 45 −74 81 99 64 HT-29 69 −66 107 101 91 Melanoma B16-F0 84 7 123 116 56 SK-MEL-5 −69 −96 95 118 88 Ovarian Cancer OVCAR-3 −33 −93 67 86 60 SK-OV-3 6 −35 82 98 74 Prostrate Cancer LNCaP −5 −86 73 84 56 PC-3 −87 70 99 64 Breast Cancer BT474 56 −67 118 108 64 MCF7 −51 −71 114 112 77 MCF-7 AdrR −43 −90 92 101 77 MDA-MB-231 −38 −92 97 100 84 MDA-MB-468 −69 −92 37 85 52 T-47D 102 20 104 119 47 4T1 75 52 60 99 26 Kidney Cancer A-498 43 −43 91 92 89 ACHN 34 −41 101 105 105 Liver Cancer HC-4 80 −91 104 105 91 Hep3B 20 −84 87 84 93 HepG2 27 −23 95 103 90 Lymphoma Ramos −94 −92 33 129 −82 H33HJ-JA1 −73 −90 57 90 −87 U937 28 −86 102 105 20 Pancreas MIA PaCa-2 20 3 92 106 92 PANC-1 64 36 87 116 82 Skin A-431 64 −89 100 102 107 A375 9 −5 74 100 65 Stomach KATO III 12 −91 84 105 83 Uterus MES-SA −57 −88 75 94 68 Cell Viability Assay Results

At 10 μM Compound 1 showed potent reduction in cell viability (−80 to −100%) towards cancer cell lines. Moderate reduction in cell viability (−40 to −80%) was observed towards 12 cancer cell lines and weak effect towards cell viability for 4 cell lines. Growth inhibition was observed for the remaining cell lines.

At 10 μM Compound 2 showed weak reduction in cell viability (−35%) towards the HL-60 leukemia cells and no significant effect on the remaining cell lines. At 10 μM Compound 3 showed no significant effect on all 44 cell lines. At 10 μM Compound 4 showed potent reduction in cell viability (−82 to −94%) towards six lymphoma, leukemia and brain cancer cell lines: Ramos and H33HJ-JA1 (lymphoma); HL-60 (TB), MOLT-4 and MV-411 (leukemia), and SK-N-MC (brain). Compound 4 was selective with no significant reduction in the growth of HUVEC endothelium cells (normal cell line).

At 10.5 μg/mL Myoporum insulare resin showed potent reduction in cell viability (−80 to −100%) towards four cancer cell lines: SW-620 (colon); Ramos (lymphoma); and MV-411 and MOLT4 (leukemia). Moderate reduction in cell viability (−40 to −80%) was observed towards six cancer cell lines: MCF-7, MCF7 AdrR, MDA-MB-468 (breast); H33HJ-JA1 (lymphoma); SK-MEL-5 (melanoma); and MES-SA (uterus). Weak cell viability reduction (−5 to −38%) towards five cell lines: K-562 (leukemia); OVCAR-3 (ovarian); LNCaP (prostrate); MDA-MB-231 (breast); and A375 (skin). No significant effect was observed towards HUVEC endothelium cells (normal cell line).

OncoPanel Cell Proliferation Assay

The OncoPanel cell proliferation assay measures the proliferation response of cancer cell lines to drug treatments through high-content fluorescence imaging or bioluminescence. Whereas the cell viability assay described above is a three-day assay, the OncoPanel assay is a 10-day assay.

Experimental Procedure

Cells were grown in RPMI 1640, 10% FBS, 2 mM L-alanyl-L-glutamine, 1 mM Na pyruvate or a special medium. Cells were seeded into 384-well plates and incubated in a humidified atmosphere of 5% CO₂ at 37° C. Compounds were added the day following cell seeding. At the same time, a time zero untreated cell plate was generated. After a 10-day incubation period, cells were fixed and stained to allow fluorescence imaging of nuclei. At 7 days post-seeding, the growth media were replaced and the plates were re-dosed with the test compound.

Compounds were serially diluted in half-log steps from the highest test concentration specified in the above table, and assayed over 10 concentrations with a maximum assay concentration of 0.1% DMSO. Automated fluorescence microscopy was carried out using a Molecular Devices ImageXpress Micro XL high-content imager, and images were collected with a 4× objective. 16-bit TIFF images were acquired and analyzed with MetaXpress 5.1.0.41 software.

Data Analysis

Cell proliferation was measured by the fluorescence intensity of an incorporated nuclear dye. The output is referred to as the relative cell count, where the measured nuclear intensity is transformed to percent of control (POC) using the following formula:

${P\; O\; C} = {\frac{I_{x}}{I_{0}} \times 100}$

Where I_(x) is the nuclear intensity at concentration x, and I₀ is the average nuclear intensity of the untreated vehicle wells.

Cellular response parameters were calculated using nonlinear regression to a sigmoidal single-site dose response model:

$y = {A + \frac{B - A}{1 + \left( {C/x} \right)^{D}}}$

Where y is a response measured at concentration x, A and B are the lower and upper limits of the response, C is the concentration at the response midpoint (EC₅₀), and D is the Hill Slope (Fallahi-Sichani, M., S. et al. 2013).

Time zero non-treated plates were used to determine the number of doublings during the assay period, using the formula:

${Doublings} = {\log_{2}\left( \frac{N}{N_{T0}} \right)}$

Where N is the cell number in untreated wells at the assay end point and NTO is the cell number at the time of compound addition.

Cell count IC₅₀ is the test compound concentration at 50% of maximal possible response. EC₅₀ is the test compound concentration at the curve inflection point or half the effective response (parameter C of the fitted curve solution). GI₅₀ is the concentration needed to reduce the observed growth by half (midway between the curve maximum and the time zero value). Activity area is an estimate of the integrated area above the curve (Barretina, J. G. et al. 2012). Activity area values range from 0-10, where a value of zero indicates no inhibition of proliferation at all concentrations, and a value of 10 indicates complete inhibition of proliferation at all concentrations. In rare instances, values <0 or >10 may be observed. In these instances, values <0 should be considered as equivalent to 0, whereas values >10 should be considered equivalent to 10.

Curve-fitting, calculations, and report generation were performed using a custom data reduction engine and MathIQ based software (AIM).

The OncoPanel cell proliferation results (OncoPanel, Eurofins PanLab, USA, across 280 human cancer cell lines) for compounds 1, 2 and 4 are shown below in Table 19. Tables 21 to 28 show representative proliferation response data (Table 21: MDA-MB-415; Table 22: RKO-AS45-1; Table 23: SW480; Table 24: 639-V; Table 25: Hs 729; Table 26: Hs 852.T; Table 27: HCT-8; Table 28: IM-9) and the cell proliferation results for Myoporum insulare resin are shown below in Table 29.

TABLE 19 OncoPanel cell proliferation results for compounds 1, 2 and 4. Compound 1 Compound 2 Compound 4 Cell Cell Cell Cell Cell Cell Cell Cell Cell Count Count Count Count Count Count Count Count Count EC₅₀ IC₅₀ GI₅₀ EC₅₀ IC₅₀ GI₅₀ EC₅₀ IC₅₀ GI₅₀ Cell Line Type (μM) (μM) (μM) (μM) (μM) (μM) (μM) (μM) (μM) 5637 Bladder 3.38 3.38 3.32 >30 >30 >30 15.20 15.20 15.10 639-V Bladder 2.01 2.01 2.01 6.54 8.00 7.98 12.00 12.00 12.00 647-V Bladder 3.12 3.12 3.11 11.30 11.30 11.20 5.99 6.00 5.99 BFTC-905 Bladder 1.79 1.79 1.78 8.03 8.03 8.01 9.70 9.70 9.68 HT-1197 Bladder 2.31 2.34 2.28 10.70 10.70 10.30 15.60 15.60 15.20 HT1376 Bladder 1.45 1.46 1.43 4.71 4.71 4.47 14.10 14.10 13.80 J82 Bladder 1.06 1.06 1.05 12.90 12.90 12.70 6.22 6.22 6.09 SCaBER Bladder 3.12 3.12 3.10 11.50 11.50 11.40 16.90 16.90 16.80 T24 Bladder 1.44 1.44 1.44 20.40 20.40 20.30 12.20 12.20 12.20 TCCSUP Bladder 1.75 1.75 1.72 8.20 8.20 7.78 8.91 8.91 8.67 UM-UC-3 Bladder 1.85 1.85 1.84 5.30 5.75 5.74 3.83 3.83 3.82 AU565 Breast 1.62 1.62 1.61 6.40 6.40 6.29 10.10 10.10 10.00 BT20 Breast 1.59 1.61 1.53 12.20 12.70 12.30 14.10 14.30 13.90 BT474 Breast 5.41 5.46 4.95 >30 >30 >30 25.50 25.70 25.00 BT-549 Breast 3.14 3.14 3.12 25.80 25.90 25.80 15.30 15.30 15.10 CAMA-1 Breast 3.51 3.51 3.40 >30 >30 >30 >30 >30 >30 EFM-19 Breast 1.61 1.61 1.54 11.20 11.20 10.60 11.90 11.90 11.30 Hs 578T Breast 4.74 4.76 4.68 28.70 28.70 28.20 15.00 15.00 14.90 KPL-1 Breast 0.81 0.81 0.81 4.70 4.73 4.70 4.18 4.18 4.13 MCF7 Breast 1.37 1.37 1.35 10.40 10.40 9.96 12.10 12.10 11.90 MDA MB 231 Breast 1.47 1.48 1.47 4.39 4.39 4.36 11.40 11.40 11.30 MDA MB 453 Breast 0.78 0.78 0.77 3.68 3.69 3.65 3.76 3.76 3.68 MDA MB 468 Breast 2.75 2.76 2.74 8.07 8.08 8.00 7.41 7.43 7.34 MDA-MB-415 Breast 1.02 1.02 1.00 11.50 11.50 10.80 28.50 28.50 27.40 MDA-MB-436 Breast 2.68 2.69 2.67 9.72 9.77 9.73 10.30 10.30 10.20 SK-BR-3 Breast 1.79 1.80 1.76 12.20 12.20 11.90 10.30 10.30 9.97 T47D Breast 4.63 4.63 4.28 10.20 10.20 9.40 3.28 3.28 2.92 A172 CNS - Glioma 1.12 1.15 1.14 9.02 9.02 8.93 8.08 8.08 8.03 CCF-STTG1 CNS - Glioma 5.79 6.60 5.24 >30 >30 >30 19.70 19.70 16.00 DBTRG-05MG CNS - Glioma 2.65 2.66 2.64 12.20 13.00 12.80 17.60 17.60 17.40 DK-MG CNS - Glioma 6.16 6.41 6.12 1.64 >30 >30 17.20 17.20 16.40 H4 CNS - Glioma 2.77 2.77 2.77 3.06 >30 >30 13.00 13.00 13.00 Hs 683 CNS - Glioma 4.90 4.91 4.76 29.80 29.80 28.30 24.50 24.60 24.30 M059J CNS - Glioma 3.09 3.09 3.02 9.98 15.80 14.20 13.00 13.00 12.90 PFSK-1 CNS - Glioma 4.32 4.33 4.31 14.70 14.70 14.60 3.82 3.82 3.80 SNB-19 CNS - Glioma 1.66 1.66 1.65 17.30 17.30 16.80 13.00 13.00 12.90 SW1088 CNS - Glioma 2.44 2.44 2.43 10.70 10.70 10.50 8.77 8.77 8.69 SW1783 CNS - Glioma 3.19 3.19 3.09 10.20 11.00 10.50 12.30 12.30 12.10 T98G CNS - Glioma 2.05 2.05 2.05 19.10 19.10 19.00 24.50 24.50 24.50 U-118 MG CNS - Glioma 4.56 4.58 4.49 29.60 29.60 29.40 13.90 13.90 13.60 U-138MG CNS - Glioma 6.29 6.33 6.15 >30 >30 >30 25.00 25.00 24.90 U-87 MG CNS - Glioma 2.12 2.12 2.04 >30 >30 >30 13.80 13.80 13.60 D341 Med CNS - 9.09 9.46 9.37 19.20 19.20 18.70 8.57 8.57 8.40 Medulloblastoma Daoy CNS - 1.06 1.06 1.06 8.64 8.65 8.64 3.19 3.19 3.19 Medulloblastoma BE(2)C CNS - 4.23 4.23 4.18 6.91 7.52 7.48 13.10 13.10 13.00 Neuroblastoma CHP-212 CNS - 1.43 1.43 1.41 11.40 11.40 11.10 9.77 9.78 9.74 Neuroblastoma MC-IXC CNS - 4.01 4.01 4.00 16.90 16.90 16.90 6.88 6.88 6.88 Neuroblastoma SK-N-AS CNS - 3.58 3.58 3.54 6.23 7.92 7.70 5.86 5.86 5.75 Neuroblastoma SK-N-DZ CNS - 3.16 3.16 2.94 >30 >30 >30 11.90 11.90 11.50 Neuroblastoma SK-N-FI CNS - 7.31 7.31 6.32 >30 >30 >30 21.50 21.50 20.60 Neuroblastoma Colo 201 Colon 0.92 0.92 0.92 8.51 8.51 8.45 4.07 4.07 4.04 Colo 205 Colon 1.31 1.31 1.31 10.70 10.70 10.70 11.90 11.90 11.90 Colo 320 HSR Colon 3.09 3.13 3.13 11.60 11.60 11.60 5.15 5.16 5.15 Colo 320DM Colon 3.20 3.20 3.18 7.72 7.72 7.67 7.91 7.91 7.87 DLD-1 Colon 2.96 2.96 2.96 10.70 11.20 11.20 15.70 15.70 15.70 HCT-116 Colon 1.75 1.75 1.75 6.68 6.68 6.67 10.30 10.30 10.30 HCT-15 Colon 1.84 1.84 1.83 8.64 9.49 9.48 13.40 13.40 13.40 HCT-8 Colon 1.23 1.23 1.23 6.27 6.27 6.24 12.90 12.90 12.90 HT-29 Colon 4.01 4.01 4.00 19.50 19.50 19.40 17.00 17.00 17.00 LS1034 Colon 1.87 1.87 1.83 12.70 12.70 12.00 23.60 23.60 23.50 LS123 Colon 1.13 1.16 1.03 6.57 18.40 13.40 12.00 12.00 10.70 LS411N Colon 8.72 8.72 8.51 23.90 23.90 23.40 23.10 23.10 22.60 MT-3 Colon 3.67 3.67 3.66 16.90 16.90 16.80 23.90 23.90 23.80 NCI-H508 Colon 14.40 14.40 14.30 >30 >30 >30 >30 >30 >30 NCI-H747 Colon 3.09 3.11 3.10 12.30 >30 >30 28.10 28.20 28.10 RKO Colon 1.69 1.69 1.69 7.22 7.92 7.92 12.00 12.00 12.00 RKO-AS45-1 Colon 1.50 1.50 1.50 9.64 9.64 9.62 12.80 12.80 12.80 RKOE6 Colon 1.72 1.72 1.72 6.95 6.95 6.93 6.79 6.79 6.76 SW1417 Colon 3.15 3.15 3.07 22.80 22.80 21.80 >30 >30 29.30 SW1463 Colon 3.73 3.75 3.71 >30 >30 >30 29.40 29.60 29.50 SW403 Colon 8.09 8.11 7.89 >30 >30 >30 >30 >30 >30 SW48 Colon 1.33 1.33 1.32 8.73 8.73 8.65 13.00 13.00 13.00 SW480 Colon 1.21 1.21 1.20 10.10 10.10 9.93 12.30 12.30 12.30 SW620 Colon 1.48 1.48 1.48 13.20 13.20 13.20 11.30 11.30 11.30 SW837 Colon 2.35 2.37 2.33 >30 >30 >30 18.90 18.90 18.70 SW948 Colon 2.21 2.21 2.20 14.70 14.70 14.50 29.70 >30 >30 WiDr Colon 2.32 2.32 2.31 14.50 14.50 14.40 12.80 12.80 12.80 NCI-H295R Endocrine - 14.70 14.70 13.50 >30 >30 >30 25.60 25.60 21.60 Adrenal gland BHT-101 Endocrine - 2.16 2.17 2.17 14.00 14.00 13.90 6.80 6.80 6.77 Thyroid CAL-62 Endocrine - 1.69 1.69 1.69 5.60 5.79 5.78 5.36 5.36 5.36 Thyroid CGTH-W-1 Endocrine - 1.20 1.20 1.20 5.83 5.83 5.81 3.45 3.48 3.47 Thyroid SW579 Endocrine - 1.71 1.71 1.69 12.30 12.30 12.20 11.00 11.00 11.00 Thyroid Y79 Eye 5.60 5.60 5.33 6.57 7.65 7.41 5.65 5.65 5.34 C-33A Female GU - 7.18 7.18 7.08 12.00 12.00 11.90 9.57 9.57 9.42 Cervix C-4 II Female GU - 1.09 1.10 1.09 8.94 9.03 9.00 12.60 12.60 12.50 Cervix HeLa Female GU - 2.99 2.99 2.98 16.60 16.60 16.50 21.10 21.10 21.10 Cervix HT-3 Female GU - 3.88 3.89 3.84 17.60 19.30 18.60 14.50 14.50 14.30 Cervix SiHa Female GU - 5.22 5.23 5.17 28.30 28.30 25.80 11.30 13.50 13.20 Cervix Ca Ski Female GU - 5.28 5.29 5.24 >30 >30 >30 17.60 17.60 17.60 Ovary CaOV3 Female GU - 1.91 1.92 1.85 10.20 10.20 9.61 10.20 10.30 10.20 Ovary ME-180 Female GU - 2.25 2.26 2.21 >30 >30 >30 25.30 25.30 25.20 Ovary MS751 Female GU - 2.23 2.23 2.21 7.55 8.78 8.72 12.10 12.10 12.00 Ovary OVCAR3 Female GU - 1.53 1.57 1.52 14.40 14.40 13.70 15.60 15.60 14.90 Ovary PA-1 Female GU - 4.21 4.21 4.21 11.60 16.30 16.20 7.89 7.90 7.89 Ovary SKOV3 Female GU - 1.58 1.58 1.56 15.40 15.40 15.20 8.43 8.43 8.33 Ovary AN3 CA Female GU - 1.52 1.52 1.49 11.30 11.30 11.10 9.62 9.62 9.43 Uterus HEC-1-A Female GU - 2.57 2.57 2.56 12.10 12.10 12.00 12.30 12.30 12.20 Uterus KLE Female GU - 3.70 3.82 3.56 26.30 26.30 20.50 18.20 18.20 16.60 Uterus SW954 Female GU - 7.63 7.63 7.60 >30 >30 >30 21.70 21.70 21.60 Vulva BV-173 Leukemia 3.80 3.80 3.79 9.33 9.33 9.33 3.56 3.56 3.55 CCRFCEM Leukemia 4.73 4.73 4.71 11.50 11.50 11.40 4.92 4.93 4.91 CEM-C1 Leukemia 1.96 1.96 1.95 3.26 3.26 3.26 1.47 1.47 1.47 CML-T1 Leukemia 1.93 1.93 1.93 3.69 3.69 3.68 3.93 3.93 3.93 EM-2 Leukemia 2.39 2.39 2.37 10.80 10.80 10.80 3.99 4.00 4.00 HEL-92-1-7 Leukemia 13.90 13.90 13.90 13.40 13.40 13.40 4.05 4.05 4.05 J-RT3-T3-5 Leukemia 0.01 0.01 0.010 2.08 2.08 2.06 2.71 2.71 2.71 Jurkat Leukemia 2.25 2.25 2.24 2.66 2.66 2.65 3.59 3.59 3.58 K562 Leukemia 4.60 4.60 4.58 10.20 10.20 10.10 12.20 12.20 12.20 KG-1 Leukemia 0.12 0.12 0.11 11.30 12.00 11.90 13.30 13.30 13.10 KU812 Leukemia 2.81 2.81 2.57 6.52 6.52 5.66 9.50 9.50 9.13 MEG01 Leukemia 7.35 7.35 7.22 13.80 13.80 13.70 8.39 8.39 8.37 MHH-PREB-1 Leukemia 6.17 6.17 6.17 7.85 7.85 7.85 5.71 5.71 5.71 MOLT-16 Leukemia 1.84 1.84 1.84 13.30 13.30 13.30 4.02 4.02 4.01 MOLT-3 Leukemia 2.13 2.13 2.11 1.27 1.27 1.26 3.15 3.15 3.13 MV-4-11 Leukemia 3.58 3.58 3.57 8.56 8.56 8.52 3.81 3.81 3.80 MX1 Leukemia 1.29 1.29 1.29 10.80 10.80 10.80 2.46 2.46 2.46 NALM-6 Leukemia 1.81 1.81 1.81 1.67 1.68 1.68 2.61 2.61 2.61 RS4; 11 Leukemia 1.77 1.77 1.71 6.43 6.43 6.30 4.15 4.23 4.16 TF-1 Leukemia 2.11 2.11 1.98 2.75 2.75 2.44 6.57 6.57 6.26 Thp1 Leukemia 1.38 1.38 1.34 5.32 5.69 5.61 12.00 12.00 11.90 BC-1 Lymphoma 1.52 1.52 1.51 7.95 7.95 7.93 4.16 4.16 4.16 BCP-1 Lymphoma 3.18 3.18 3.14 8.48 8.48 8.40 8.82 8.82 8.75 CA46 Lymphoma 2.27 2.27 2.26 3.83 3.83 3.82 4.06 4.06 4.06 CRO-AP2 Lymphoma 5.72 5.72 5.66 6.38 6.38 6.36 9.94 9.94 9.88 Daudi Lymphoma 1.84 1.84 1.84 3.93 3.93 3.92 2.28 2.28 2.28 DB Lymphoma 2.10 2.10 2.09 3.23 3.25 3.24 3.85 3.85 3.84 DOHH-2 Lymphoma 1.41 1.41 1.41 3.24 3.24 3.24 2.24 2.24 2.24 DoTc2 4510 Lymphoma 1.03 1.03 1.01 6.07 6.07 5.84 9.23 9.23 9.09 EB2 Lymphoma 6.47 6.49 6.45 23.30 23.30 23.00 8.35 8.38 8.32 EB-3 Lymphoma 3.56 3.56 3.56 9.72 9.72 9.71 3.93 3.93 3.93 GA-10 Lymphoma 1.93 1.94 1.94 3.72 3.73 3.73 1.23 1.23 1.23 Hs 445 Lymphoma 1.30 1.32 1.31 4.34 4.34 4.23 5.80 5.80 5.74 Hs 611.T Lymphoma 2.92 2.94 2.91 10.70 10.70 10.60 5.11 5.11 5.05 HT Lymphoma 1.52 1.53 1.51 6.51 6.51 6.43 4.49 4.49 4.46 JeKo-1 Lymphoma 5.23 5.23 5.21 4.48 4.48 4.47 3.91 3.91 3.91 Jiyoye Lymphoma 5.88 5.88 5.87 17.80 17.80 17.70 3.55 3.55 3.55 L-428 Lymphoma 1.08 1.09 1.08 8.87 8.87 8.83 2.07 2.07 2.06 MC116 Lymphoma 3.79 3.79 3.78 13.80 13.80 13.80 12.50 12.50 12.40 NAMALWA Lymphoma 3.39 3.39 3.39 5.71 5.71 5.70 3.91 3.91 3.90 Raji Lymphoma 4.60 4.60 4.59 8.04 8.04 8.04 3.92 3.92 3.92 Ramos (RA 1) Lymphoma 2.96 2.96 2.96 4.39 4.39 4.39 4.17 4.17 4.17 RPMI 6666 Lymphoma 3.53 3.53 3.44 10.40 10.70 10.70 23.10 23.10 23.00 SR Lymphoma 3.80 3.80 3.80 10.70 10.70 10.70 4.39 4.39 4.39 ST486 Lymphoma 1.09 1.09 1.08 3.87 3.87 3.86 2.66 2.66 2.65 SU-DHL-10 Lymphoma 3.94 3.94 3.93 9.92 9.92 9.90 7.68 7.68 7.67 SU-DHL-4 Lymphoma 2.01 2.01 2.01 4.20 4.20 4.20 5.74 5.74 5.74 SU-DHL-5 Lymphoma 1.20 1.20 1.19 4.28 4.28 4.27 3.80 3.80 3.80 SU-DHL-8 Lymphoma 1.77 1.77 1.77 7.40 7.40 7.40 3.90 3.90 3.90 SUP-T1 Lymphoma 3.96 3.96 3.95 12.10 12.10 12.00 6.51 6.51 6.49 TUR Lymphoma 5.09 5.09 5.08 2.63 >30 >30 8.98 8.98 8.98 ARH-77 Myeloma 1.95 1.97 1.95 9.50 9.60 9.57 13.50 13.50 13.40 IM-9 Myeloma 0.42 0.42 0.42 3.92 3.92 3.92 6.71 6.71 6.71 RPMI 8226 Myeloma 0.68 0.68 0.67 2.02 2.02 1.95 3.03 3.04 3.03 SKO-007 Myeloma 0.99 0.99 0.93 1.59 1.59 1.42 4.66 4.71 4.55 U266B1 Myeloma 3.22 3.22 3.08 9.15 9.28 9.01 10.10 10.10 9.81 A-253 Head and Neck 4.13 4.14 4.13 28.50 28.70 28.70 18.20 18.20 18.10 A388 Head and Neck 1.66 1.67 1.66 13.50 13.50 13.40 7.66 7.66 7.59 A431 Head and Neck 2.80 2.81 2.80 >30 >30 >30 24.80 24.80 24.80 Cal 27 Head and Neck 2.16 2.16 2.15 7.64 7.64 7.62 12.30 12.30 12.20 Detroit 562 Head and Neck 1.96 1.97 1.96 14.50 14.50 14.40 25.00 25.00 25.00 FaDu Head and Neck 0.87 0.87 0.87 8.66 8.66 8.66 8.28 8.28 8.28 OE19 Head and Neck 3.23 3.23 3.21 >30 >30 >30 20.70 20.70 20.50 OE21 Head and Neck 3.68 3.68 3.67 >30 >30 >30 12.70 12.70 12.70 SCC-25 Head and Neck 3.94 3.96 3.95 >30 >30 >30 25.00 25.00 25.00 SCC-4 Head and Neck 4.66 4.72 4.63 >30 >30 >30 15.80 15.80 15.50 SCC-9 Head and Neck 3.44 3.45 3.44 26.70 27.30 27.30 16.50 16.50 16.40 769-P Kidney 0.85 0.85 0.85 6.95 6.95 6.94 3.72 3.72 3.71 786-O Kidney 2.05 2.05 2.05 27.90 27.90 27.90 14.00 14.00 13.90 A498 Kidney 1.29 1.29 1.29 13.30 13.30 13.20 12.30 12.30 12.20 A-704 Kidney 1.57 1.60 1.54 >30 >30 >30 5.70 5.70 5.41 ACHN Kidney 1.74 1.74 1.74 8.79 8.79 8.76 9.50 9.50 9.47 Caki-1 Kidney 2.86 2.86 2.75 0.17 >30 >30 14.40 14.40 14.10 Caki-2 Kidney 4.38 4.38 4.35 28.80 29.10 28.90 13.10 13.60 13.50 G-401 Kidney 4.00 4.00 3.99 26.00 26.20 26.20 8.08 8.08 8.05 SK-NEP-1 Kidney 0.01 0.01 0.006 0.06 0.06 0.06 9.62 9.63 9.62 HepG2 Liver 0.82 0.82 0.81 6.74 6.95 6.86 25.60 25.70 25.60 HLE Liver 4.62 4.63 4.59 10.00 16.40 16.00 7.24 7.24 7.15 HLF Liver 4.47 4.48 4.45 13.70 15.70 15.60 8.72 8.74 8.70 HuCCT1 Liver 2.48 2.48 2.48 22.60 22.60 22.60 24.80 24.80 24.80 HUH-6 Clone 5 Liver 1.52 1.52 1.51 15.50 15.50 15.30 9.63 9.63 9.46 OCUG-1 Liver 4.30 4.30 4.29 >30 >30 29.90 15.60 15.60 15.50 SNU-423 Liver 1.48 1.49 1.46 5.29 5.31 5.26 9.30 9.31 9.28 A427 Lung - NSCLC 4.52 4.52 4.50 11.20 11.20 11.20 10.00 10.00 9.98 A549 Lung - NSCLC 1.66 1.67 1.66 22.00 22.00 21.90 10.90 10.90 10.80 Calu1 Lung - NSCLC 3.10 3.10 3.07 13.90 13.90 13.80 8.88 8.88 8.77 Calu6 Lung - NSCLC 9.24 9.24 8.59 14.50 16.60 15.70 25.00 25.00 24.50 ChaGoK1 Lung - NSCLC 1.53 1.54 1.53 8.32 8.32 8.26 8.92 8.92 8.82 COR-L105 Lung - NSCLC 1.44 1.45 1.42 4.76 4.76 4.61 8.66 8.66 8.47 COR-L23 Lung - NSCLC 7.18 7.18 7.14 23.80 23.80 23.60 13.30 13.30 13.20 Hs 229.T Lung - NSCLC 2.88 2.94 2.89 29.30 29.80 28.60 25.90 26.30 25.20 NCI-H292 Lung - NSCLC 3.20 3.20 3.18 16.70 16.70 16.60 9.21 9.21 9.19 NCIH441 Lung - NSCLC 12.10 12.10 11.70 >30 >30 >30 4.76 >30 >30 NCI-H460 Lung - NSCLC 2.34 2.34 2.33 5.57 5.92 5.91 12.20 13.00 13.00 NCI-H520 Lung - NSCLC 2.43 2.43 2.41 9.03 9.20 9.17 20.80 20.80 20.60 NCI-H596 Lung - NSCLC 8.78 8.78 8.41 12.00 15.10 14.40 28.20 >30 >30 NCI-H661 Lung - NSCLC 2.28 2.29 2.28 9.51 9.51 9.51 5.94 5.94 5.86 SKMES1 Lung - NSCLC 1.06 1.06 1.06 7.80 7.80 7.67 11.60 11.60 11.50 DMS114 Lung - SCLC 2.77 2.77 2.57 11.50 13.80 13.10 11.50 11.60 11.40 DMS53 Lung - SCLC 0.81 0.81 0.77 5.91 5.95 5.75 8.95 8.95 8.60 NCIH446 Lung - SCLC 10.30 10.30 10.00 9.75 9.85 9.77 7.81 7.81 7.62 NCI-H69 Lung - SCLC 6.30 6.44 5.84 16.20 16.20 14.90 15.90 15.90 15.10 SHP-77 Lung - SCLC 0.89 0.90 0.89 8.01 8.01 7.90 6.83 6.83 6.75 SW900 Lung - SCLC 3.29 3.33 3.24 26.60 26.80 26.70 23.30 23.30 23.10 AsPC-1 Pancreas 2.87 2.87 2.84 14.10 14.10 13.80 14.70 14.70 14.50 BxPC-3 Pancreas 3.29 3.30 3.29 13.00 >30 >30 13.40 13.40 13.40 Capan-1 Pancreas >30 >30 >30 >30 >30 >30 >30 >30 >30 Capan-2 Pancreas 1.33 1.33 1.32 8.64 8.64 8.48 24.80 24.80 24.70 CFPAC-1 Pancreas 3.65 3.66 3.65 12.30 13.50 13.40 25.20 25.20 25.10 HPAF-II Pancreas 1.38 1.38 1.37 9.46 9.62 9.60 12.40 12.50 12.40 Hs 766T Pancreas 1.61 1.61 1.54 28.40 28.40 26.20 9.34 9.34 9.08 HuP-T4 Pancreas 4.52 4.54 4.53 >30 >30 >30 13.70 13.70 13.60 Mia PaCa-2 Pancreas 2.97 2.97 2.97 16.30 16.30 16.20 11.00 11.00 11.00 PANC-1 Pancreas 2.87 2.87 2.83 7.06 7.06 7.01 11.70 11.70 11.60 PSN-1 Pancreas 2.32 2.32 2.32 12.00 12.00 12.00 5.04 5.04 5.04 SU.86.86 Pancreas 7.61 7.63 7.58 13.10 >30 >30 >30 >30 >30 YAPC Pancreas 1.91 1.91 1.90 13.00 13.00 12.90 18.00 18.20 18.20 BeWo Placenta 9.06 9.06 8.92 7.68 >30 >30 15.60 15.60 15.50 JAR Placenta 12.20 12.20 12.20 >30 >30 >30 6.53 6.53 6.49 JEG-3 Placenta 4.33 4.33 4.32 >30 >30 >30 6.50 6.51 6.48 22Rv1 Prostate 4.08 4.08 4.06 11.40 11.40 11.30 9.08 9.08 9.00 BM-1604 Prostate 2.58 2.58 2.56 19.10 19.10 18.90 >30 >30 >30 BPH1 Prostate 5.24 5.24 5.20 >30 >30 >30 17.30 17.30 17.30 DU145 Prostate 2.80 2.80 2.78 21.00 21.00 20.70 18.10 18.10 18.10 LNCaP Prostate 3.90 3.90 3.47 0.86 0.86 0.28 11.50 12.90 12.40 PC-3 Prostate 3.88 3.88 3.86 12.00 12.00 11.80 7.71 7.71 7.64 A101D Skin (Melanoma) 8.42 8.42 8.25 >30 >30 >30 >30 >30 >30 A375 Skin (Melanoma) 1.05 1.05 1.05 6.45 6.45 6.44 5.74 5.74 5.74 A7 Skin (Melanoma) 0.94 0.94 0.94 5.54 5.63 5.60 10.50 10.50 10.50 C32 Skin (Melanoma) 2.11 2.11 1.81 19.20 19.20 15.50 7.84 7.84 6.37 CHL-1 Skin (Melanoma) 1.37 1.37 1.36 14.30 14.30 14.20 11.90 11.90 11.80 COLO 829 Skin (Melanoma) 0.68 0.68 0.62 5.35 5.35 4.93 3.71 3.71 3.29 G-361 Skin (Melanoma) 1.79 1.79 1.77 11.70 11.80 11.80 13.80 13.90 13.80 HMCB Skin (Melanoma) 1.37 1.37 1.36 6.68 6.68 6.57 9.49 9.49 9.38 Hs 294T Skin (Melanoma) 2.64 2.64 2.59 7.01 7.01 6.72 8.33 8.33 8.23 Hs 688(A).T Skin (Melanoma) 1.33 1.44 1.27 11.60 13.20 12.20 7.43 7.68 6.88 Hs 695T Skin (Melanoma) 1.18 1.27 1.16 9.07 9.48 8.99 5.00 5.18 4.02 Hs 852.T Skin (Melanoma) 2.49 2.56 2.42 15.40 15.40 14.70 12.00 12.00 11.60 Hs 934.T Skin (Melanoma) 2.53 3.27 2.34 >30 >30 24.10 11.10 11.60 10.20 Hs 936.T(C1) Skin (Melanoma) 2.00 2.00 1.97 3.62 3.76 3.74 9.10 10.40 10.20 MALME3M Skin (Melanoma) 1.79 1.79 1.66 19.80 19.80 17.30 20.60 20.60 17.80 MeWo Skin (Melanoma) 1.32 1.32 1.31 9.45 9.97 9.91 9.66 9.66 9.51 RPMI-7951 Skin (Melanoma) 0.70 0.70 0.70 5.77 6.15 6.13 9.98 9.98 9.96 SH-4 Skin (Melanoma) 1.95 1.95 1.94 9.60 11.90 11.80 11.60 11.60 11.50 SK-MEL-1 Skin (Melanoma) 4.29 4.41 4.11 15.70 15.70 14.70 >30 >30 >30 SK-MEL-28 Skin (Melanoma) 1.54 1.54 1.53 9.46 9.89 9.85 11.20 11.20 11.10 SK-MEL-3 Skin (Melanoma) 4.37 4.38 4.28 22.00 22.00 20.80 >30 >30 >30 WM-266-4 Skin (Melanoma) 1.15 1.15 1.14 6.39 6.39 6.25 10.90 10.90 10.80 G-292, clone Soft Tissue - 4.55 4.57 4.41 8.80 8.80 8.57 6.23 6.25 6.07 A141B1 Osteosarcoma HOS Soft Tissue - 0.96 0.96 0.96 4.82 4.82 4.81 3.08 3.08 3.08 Osteosarcoma Hs 888.Sk Soft Tissue - 2.20 2.38 2.05 28.30 28.50 27.90 12.30 12.70 12.10 Osteosarcoma KHOS-240S Soft Tissue - 0.95 0.95 0.95 4.59 4.59 4.58 4.96 4.96 4.96 Osteosarcoma MG-63 Soft Tissue - 9.24 9.24 9.16 >30 >30 >30 11.20 11.20 11.10 Osteosarcoma SaOS2 Soft Tissue - 1.98 2.10 2.04 15.70 15.70 15.10 10.60 10.70 10.70 Osteosarcoma SJSA1 Soft Tissue - 1.41 1.41 1.41 7.11 7.11 7.02 7.45 7.45 7.45 Osteosarcoma SW1353 Soft Tissue - 1.00 1.00 0.99 9.49 9.49 9.46 4.15 4.15 4.14 Osteosarcoma U2OS Soft Tissue - 1.66 1.66 1.65 12.70 12.70 12.70 4.21 4.21 4.20 Osteosarcoma A204 Soft Tissue - 0.52 0.54 0.52 8.46 8.54 8.50 0.72 0.72 0.68 Sarcoma A-673 Soft Tissue - 1.54 1.55 1.54 10.80 11.10 11.10 2.88 2.88 2.87 Sarcoma Hs 729 Soft Tissue - 2.77 2.78 2.67 23.30 23.30 21.80 11.90 11.90 11.60 Sarcoma Hs 821.T Soft Tissue - 4.22 4.22 1.80 >30 >30 13.90 12.60 12.90 9.99 Sarcoma HT-1080 Soft Tissue - 0.96 0.96 0.96 3.31 3.31 3.30 4.42 4.42 4.42 Sarcoma MES-SA Soft Tissue - 1.23 1.23 1.22 9.26 9.26 9.22 6.49 6.49 6.44 Sarcoma RD Soft Tissue - 0.81 0.81 0.81 2.98 2.98 2.96 5.19 5.19 5.17 Sarcoma SJRH30 Soft Tissue - 1.42 1.42 1.41 5.09 5.10 5.07 3.68 3.68 3.68 Sarcoma SK-LMS-1 Soft Tissue - 1.57 1.57 1.56 6.79 11.30 11.20 9.04 9.04 8.98 Sarcoma SK-UT-1 Soft Tissue - 2.23 2.23 2.22 11.70 11.70 11.60 5.49 5.49 5.46 Sarcoma SW684 Soft Tissue - 9.37 9.37 8.28 >30 >30 >30 >30 >30 >30 Sarcoma SW872 Soft Tissue - 0.96 0.96 0.96 4.46 4.46 4.45 4.39 4.39 4.38 Sarcoma SW982 Soft Tissue - 1.07 1.07 1.07 7.63 7.63 7.53 7.29 7.30 7.26 Sarcoma TE 125.T Soft Tissue - 6.47 9.21 1.48 >30 >30 28.70 23.60 23.60 17.00 Sarcoma TE 381.T Soft Tissue - 1.62 1.62 1.59 8.14 8.14 7.99 6.28 6.28 6.08 Sarcoma VA-ES-BJ Soft Tissue - 3.67 3.67 3.55 >30 >30 >30 15.20 15.20 15.00 Sarcoma AGS Stomach 0.70 0.70 0.70 4.69 4.69 4.69 5.25 5.25 5.24 HS 746T Stomach 5.58 5.74 4.64 >30 >30 >30 20.00 20.00 17.10 KATO III Stomach 3.28 3.28 3.27 8.98 16.80 16.60 10.90 11.10 11.10 SK-PN-DW Stomach 4.66 4.66 4.63 10.80 10.90 10.90 4.91 4.91 4.88 SNU-1 Stomach 1.25 1.25 1.25 8.51 9.42 9.41 6.99 6.99 6.98 SNU-16 Stomach 1.31 1.31 1.31 9.96 9.96 9.91 11.00 11.00 11.00 SNU-5 Stomach 3.92 3.92 3.87 >30 >30 >30 14.40 14.40 14.20 NTERA-2 cl.D1 Testis 2.25 2.25 2.20 12.50 12.50 12.30 7.41 7.41 7.39

TABLE 20 Oncopanel cell proliferation results for Compounds 11 and 12. Compound 11 and 12 Cell Cell Cell Count Count Count EC₅₀ IC₅₀ GI₅₀ Cell Line Type (μM) (μM) (μM) 647-V Bladder >30 >30 >30 AU565 Breast 16.4 17.1 16.9 CAMA-1 Breast >30 >30 >30 MCF7 Breast 27.5 27.5 27 MDA MB 231 Breast >30 >30 >30 MDA MB 453 Breast 10.6 10.6 10.4 MDA-MB-415 Breast 17.8 17.8 15.7 Hs 683 CNS - Glioma 27 27 25.6 M059J CNS - Glioma >30 >30 >30 HuTu 80 Colon 8.61 8.7 8.65 LS-174T Colon >30 >30 >30 HeLa Female GU - Cervix >30 >30 >30 OVCAR3 Female GU - Ovary >30 >30 >30 AN3 CA Female GU - Uterus 10.8 13.3 13.1 MEG01 Leukemia 17.5 17.5 17.3 MOLT-16 Leukemia 21.8 21.8 21.7 MOLT-3 Leukemia 11.8 14.1 14 MV-4-11 Leukemia >30 >30 >30 MX1 Leukemia 14.9 14.9 14.9 A427 Lung - NSCLC 16 16 15.8 Calu1 Lung - NSCLC >30 >30 >30 Calu6 Lung - NSCLC >30 >30 >30 Hs 229.T Lung - NSCLC >30 >30 >30 NCI-H292 Lung - NSCLC 12.1 >30 >30 NCI-H520 Lung - NSCLC >30 >30 >30 NCI-H596 Lung - NSCLC >30 >30 >30 DOHH-2 Lymphoma 12.2 13.4 13.4 EB2 Lymphoma >30 >30 >30 Jiyoye Lymphoma >30 >30 >30 L-428 Lymphoma 21.3 21.3 21.1 NAMALWA Lymphoma >30 >30 >30 Raji Lymphoma >30 >30 >30 SUP-T1 Lymphoma >30 >30 >30 PSN-1 Pancreas >30 >30 >30 Hs 934.T Skin (Melanoma) 21.9 >30 16.3 HOS Soft Tissue - Osteosarcoma >30 >30 >30 KHOS-240S Soft Tissue - Osteosarcoma >30 >30 >30 SW1353 Soft Tissue - Osteosarcoma >30 >30 >30 Hs 729 Soft Tissue - Sarcoma 26.4 >30 >30 HT-1080 Soft Tissue - Sarcoma 12.7 12.7 12.7

TABLE 21 Representative data for proliferation response when cell line MDA-MB-415 is treated with Compound 1 Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 90.2 1.3 3.02E−03 100.0 6.4 9.53E−03 98.0 8.7 3.01E−02 93.4 2.7 9.52E−02 98.1 10.3 3.01E−01 100.2 8.6 9.51E−01 62.3 5.3 3.00E+00 1.3 0.2 9.49E+00 0.3 0.0 3.00E+01 0.2 0.0

TABLE 22 Representative data for proliferation response when cell line RKO-AS45-1 is treated with Compound 1 Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 95.4 2.0 3.02E−03 83.7 7.7 9.53E−03 88.3 3.7 3.01E−02 86.2 5.6 9.52E−02 80.5 4.7 3.01E−01 90.0 6.9 9.51E−01 72.1 9.7 3.00E+00 7.6 3.5 9.49E+00 0.1 0.0 3.00E+01 0.1 0.0

TABLE 23 Representative data for proliferation response when cell line SW480 is treated with Compound 1 Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 111.8 7.0 3.02E−03 116.5 19.3 9.53E−03 103.7 9.4 3.01E−02 99.5 0.5 9.52E−02 94.1 10.2 3.01E−01 93.5 6.4 9.51E−01 69.9 17.0 3.00E+00 4.1 4.6 9.49E+00 0.2 0.0 3.00E+01 0.4 0.4

TABLE 24 Representative data for proliferation response when cell line 639-V is treated with Compound 1 Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 92.0 14.3 3.02E−03 96.1 1.9 9.53E−03 101.2 5.0 3.01E−02 103.6 8.6 9.52E−02 91.6 9.6 3.01E−01 92.6 14.1 9.51E−01 91.9 12.2 3.00E+00 15.8 4.2 9.49E+00 0.3 0.1 3.00E+01 0.0 0.0

TABLE 25 Representative data for proliferation response when cell line Hs 729 is treated with Compound 1 Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 100.6 7.6 3.02E−03 103.0 10.5 9.53E−03 108.7 4.1 3.01E−02 107.0 2.1 9.52E−02 99.9 0.7 3.01E−01 99.4 7.7 9.51E−01 98.9 1.2 3.00E+00 46.0 8.8 9.49E+00 3.2 0.2 3.00E+01 1.0 0.3

TABLE 26 Representative data for proliferation response when cell line Hs 852.T is treated with Compound 1 Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 104.3 9.1 3.02E−03 104.5 5.1 9.53E−03 100.2 6.2 3.01E−02 94.5 5.6 9.52E−02 92.2 2.4 3.01E−01 95.3 2.9 9.51E−01 85.5 4.3 3.00E+00 41.6 5.1 9.49E+00 13.1 1.7 3.00E+01 2.7 0.5

TABLE 27 Representative data for proliferation response when cell line HCT-8 is treated with Compound 1 Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 102.6 3.7 3.02E−03 104.1 9.7 9.53E−03 91.0 10.1 3.01E−02 91.8 3.5 9.52E−02 100.8 2.6 3.01E−01 94.9 4.8 9.51E−01 72.6 2.3 3.00E+00 1.9 0.5 9.49E+00 0.0 0.0 3.00E+01 0.0 0.0

TABLE 28 Representative data for proliferation response when cell line IM-9 is treated with Compound 1 Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 94.9 34.7 3.02E−03 102.8 9.5 9.53E−03 103.1 2.4 3.01E−02 107.3 9.3 9.52E−02 97.7 3.4 3.01E−01 97.5 8.6 9.51E−01 0.0 0.0 3.00E+00 0.1 0.0 9.49E+00 0.1 0.1 3.00E+01 0.0 0.0

TABLE 29 Oncopanel cell proliferation results for Myoporum insulare resin extract. M. insulare resin extract Cell Cell Cell Count Count Count EC₅₀ IC₅₀ GI₅₀ Cell Line Type (μg/mL) (μg/mL) (μg/mL) 5637 Bladder 3.71 3.75 3.75 ^(A) 639-V Bladder 3.18 3.18 3.17 ^(B) 647-V Bladder 2.69 2.69 2.69 ^(C) BFTC-905 Bladder 3.28 3.28 3.28 ^(B) HT-1197 Bladder 3.18 3.20 3.17 ^(C) HT1376 Bladder 1.31 1.31 1.27 ^(C) J82 Bladder 1.26 1.26 1.26 ^(D) SCaBER Bladder 2.74 2.74 2.73 ^(C) T24 Bladder 1.70 1.70 1.70 ^(A) TCCSUP Bladder 2.76 2.77 2.73 ^(B) UM-UC-3 Bladder 0.98 0.98 0.97 ^(C) AU565 Breast 1.62 1.62 1.61 ^(A) BT20 Breast 1.57 1.61 1.50 ^(B) BT474 Breast 5.60 5.60 4.94 ^(A) BT-549 Breast 3.25 3.25 3.16 ^(A) CAMA-1 Breast 3.57 3.57 3.50 ^(A) EFM-19 Breast 1.59 1.59 1.50 ^(A) Hs 578T Breast 8.96 9.00 8.96 ^(A) KPL-1 Breast 0.61 0.61 0.61 ^(A) MCF7 Breast 1.72 1.73 1.71 ^(C) MDA MB 231 Breast 2.03 2.03 2.03 ^(A) MDA MB 453 Breast 0.53 0.53 0.52 ^(A) MDA MB 468 Breast 2.86 2.86 2.84 ^(A) MDA-MB-415 Breast 1.12 1.12 1.05 ^(A) MDA-MB-436 Breast 2.70 2.70 2.67 ^(A) SK-BR-3 Breast 2.15 2.16 2.10 ^(C) T47D Breast 4.80 4.80 4.59 ^(B) A172 CNS - Glioma 1.46 1.46 1.45 ^(B) CCF-STTG1 CNS - Glioma 9.00 9.00 7.35 ^(C) DBTRG- CNS - Glioma 3.23 3.24 3.22 ^(C) 05MG DK-MG CNS - Glioma 5.99 6.23 6.06 ^(C) H4 CNS - Glioma 3.32 3.32 3.32 ^(C) Hs 683 CNS - Glioma 5.01 5.01 4.87 ^(B) M059J CNS - Glioma 4.62 4.62 4.55 ^(B) PFSK-1 CNS - Glioma 3.89 3.89 3.85 ^(B) SNB-19 CNS - Glioma 1.39 1.41 1.41 ^(C) SW1088 CNS - Glioma 1.79 1.79 1.78 ^(C) SW1783 CNS - Glioma 3.06 3.06 2.96 ^(B) T98G CNS - Glioma 3.06 3.06 3.06 ^(B) U-118 MG CNS - Glioma 4.20 4.24 4.20 ^(B) U-138MG CNS - Glioma 5.92 5.92 5.74 ^(C) U-87 MG CNS - Glioma 2.46 2.50 2.45 ^(C) D341 Med CNS - 3.78 3.96 3.92 ^(B) Medulloblastoma Daoy CNS - 1.10 1.11 1.10 ^(A) Medulloblastoma BE(2)C CNS - 3.07 3.07 3.05 ^(B) Neuroblastoma CHP-212 CNS - 1.19 1.19 1.14 ^(B) Neuroblastoma MC-IXC CNS - 3.32 3.32 3.30 ^(B) Neuroblastoma SK-N-AS CNS - 3.18 3.20 3.19 ^(A) Neuroblastoma SK-N-DZ CNS - 3.35 3.42 3.39 ^(B) Neuroblastoma SK-N-FI CNS - 3.37 5.39 4.66 ^(C) Neuroblastoma Colo 201 Colon 1.08 1.08 1.08 ^(D) Colo 205 Colon 1.46 1.46 1.46 ^(D) Colo 320 HSR Colon 2.45 2.45 2.44 ^(B) Colo 320DM Colon 3.19 3.19 3.17 ^(B) DLD-1 Colon 3.19 3.19 3.19 ^(A) HCT-116 Colon 1.44 1.44 1.44 ^(B) HCT-15 Colon 1.92 1.92 1.92 ^(C) HCT-8 Colon 2.56 2.56 2.55 ^(D) HT-29 Colon 4.31 4.31 4.31 ^(A) LS1034 Colon 1.96 1.96 1.93 ^(B) LS123 Colon 1.28 1.38 1.29 ^(B) LS411N Colon 6.41 6.41 6.30 ^(B) MT-3 Colon 4.10 4.10 4.10 ^(A) NCI-H508 Colon >10.5 >10.5 >10.5 ^(B) NCI-H747 Colon 3.64 3.71 3.68 ^(B) RKO Colon 1.88 1.88 1.87 ^(C) RKO-AS45-1 Colon 2.78 2.78 2.78 ^(C) RKOE6 Colon 2.49 2.49 2.49 ^(B) SW1417 Colon 3.27 4.03 3.92 ^(D) SW1463 Colon 3.64 3.68 3.64 ^(B) SW403 Colon 9.10 9.42 9.31 ^(B) SW48 Colon 1.14 1.14 1.14 ^(C) SW480 Colon 1.04 1.04 1.03 ^(C) SW620 Colon 2.32 2.32 2.32 ^(B) SW837 Colon 2.92 3.20 3.14 ^(C) SW948 Colon >10.5 >10.5 >10.5 ^(D) WiDr Colon 1.12 1.12 1.11 ^(B) NCI-H295R Endocrine - >10.5 >10.5 >10.5 ^(A) Adrenal gland BHT-101 Endocrine - 2.72 2.72 2.71 ^(C) Thyroid CAL-62 Endocrine - 1.18 1.18 1.18 ^(A) Thyroid CGTH-W-1 Endocrine - 1.03 1.03 1.03 ^(B) Thyroid SW579 Endocrine - 3.01 3.01 3.00 ^(C) Thyroid Y79 Eye 3.08 3.10 3.02 ^(D) C-33A Female GU - 4.27 4.27 4.24 ^(C) Cervix C-4 II Female GU - 1.19 1.19 1.19 ^(B) Cervix HeLa Female GU - 3.75 3.75 3.71 ^(C) Cervix HT-3 Female GU - 3.99 4.03 3.99 ^(B) Cervix SiHa Female GU - 5.60 5.60 5.53 ^(B) Cervix Ca Ski Female GU - 5.53 5.53 5.46 ^(A) Ovary CaOV3 Female GU - 2.59 2.60 2.52 ^(B) Ovary ME-180 Female GU - 3.22 3.22 3.20 ^(B) Ovary MS751 Female GU - 2.65 2.65 2.63 ^(B) Ovary OVCAR3 Female GU - 2.01 2.08 2.02 ^(D) Ovary PA-1 Female GU - 4.73 4.73 4.73 ^(B) Ovary SKOV3 Female GU - 1.73 1.73 1.72 ^(B) Ovary AN3 CA Female GU - 1.61 1.61 1.58 ^(B) Uterus HEC-1-A Female GU - 1.60 1.68 1.67 ^(A) Uterus KLE Female GU - 3.28 3.28 2.92 ^(C) Uterus SW954 Female GU - 8.51 8.51 8.47 ^(C) Vulva BV-173 Leukemia 2.17 2.17 2.17 ^(B) CCRFCEM Leukemia 3.85 3.85 3.82 ^(A) CEM-C1 Leukemia 1.13 1.13 1.12 ^(A) CML-T1 Leukemia 1.43 1.43 1.43 ^(A) EM-2 Leukemia 2.58 2.58 2.56 ^(A) HEL-92-1-7 Leukemia 5.29 5.29 5.25 ^(A) J-RT3-T3-5 Leukemia 1.29 1.29 1.29 ^(D) Jurkat Leukemia 0.91 0.91 0.91 ^(B) K562 Leukemia 3.75 3.75 3.75 ^(A) KG-1 Leukemia 4.73 5.39 5.32 ^(B) KU812 Leukemia 4.52 4.52 4.45 ^(C) MEG01 Leukemia 4.55 4.55 4.55 ^(A) MHH-PREB-1 Leukemia 3.82 3.82 3.82 ^(C) MOLT-16 Leukemia 2.49 2.49 2.48 ^(A) MOLT-3 Leukemia 1.42 1.42 1.42 ^(C) MV-4-11 Leukemia 2.76 2.76 2.75 ^(A) MX1 Leukemia 1.28 1.28 1.28 ^(C) NALM-6 Leukemia 1.03 1.03 1.03 ^(C) RS4; 11 Leukemia 1.86 1.86 1.82 ^(A) TF-1 Leukemia 4.55 4.55 4.45 ^(C) Thp1 Leukemia 1.78 1.78 1.74 ^(A) BC-1 Lymphoma 1.73 1.73 1.73 ^(A) BCP-1 Lymphoma 3.26 3.26 3.23 ^(A) CA46 Lymphoma 2.03 2.03 2.03 ^(A) CRO-AP2 Lymphoma 3.99 3.99 3.99 ^(A) Daudi Lymphoma 1.33 1.33 1.33 ^(A) DB Lymphoma 1.09 1.10 1.10 ^(B) DOHH-2 Lymphoma 1.01 1.01 1.01 ^(A) DoTc2 4510 Lymphoma 1.38 1.38 1.36 ^(A) EB2 Lymphoma 5.60 5.60 5.57 ^(A) EB-3 Lymphoma 1.68 1.68 1.68 ^(B) GA-10 Lymphoma 1.42 1.42 1.42 ^(A) Hs 445 Lymphoma 1.46 1.46 1.44 ^(C) Hs 611.T Lymphoma 3.29 3.29 3.28 ^(D) HT Lymphoma 1.61 1.61 1.60 ^(c) JeKo-1 Lymphoma 2.58 2.58 2.57 ^(A) Jiyoye Lymphoma 4.24 4.24 4.24 ^(A) L-428 Lymphoma 1.24 1.24 1.24 ^(A) MC116 Lymphoma 3.15 3.15 3.15 ^(A) NAMALWA Lymphoma 1.68 1.69 1.68 ^(D) Raji Lymphoma 3.92 3.92 3.92 ^(A) Ramos (RA 1) Lymphoma 2.33 2.33 2.33 ^(C) RPMI 6666 Lymphoma 3.28 3.32 3.27 ^(C) SR Lymphoma 1.74 1.74 1.74 ^(A) ST486 Lymphoma 1.08 1.08 1.08 ^(A) SU-DHL-10 Lymphoma 4.17 4.17 4.17 ^(A) SU-DHL-4 Lymphoma 1.82 1.82 1.82 ^(C) SU-DHL-5 Lymphoma 1.02 1.02 1.02 ^(B) SU-DHL-8 Lymphoma 1.74 1.74 1.74 ^(A) SUP-T1 Lymphoma 3.75 3.75 3.75 ^(A) TUR Lymphoma 4.48 4.48 4.48 ^(C) ARH-77 Myeloma 2.66 2.67 2.65 ^(A) IM-9 Myeloma 0.68 0.68 0.68 ^(B) RPMI 8226 Myeloma 0.99 1.00 0.99 ^(C) SKO-007 Myeloma 0.99 1.00 0.99 ^(B) U266B1 Myeloma 2.82 2.82 2.71 ^(B) A-253 Head and Neck 8.23 8.23 8.23 ^(B) A388 Head and Neck 1.92 1.94 1.93 ^(B) A431 Head and Neck 3.29 3.30 3.30 ^(A) Cal 27 Head and Neck 2.17 2.17 2.17 ^(B) Detroit 562 Head and Neck 2.99 3.00 2.99 ^(B) FaDu Head and Neck 0.90 0.90 0.90 ^(B) OE19 Head and Neck 2.97 2.98 2.96 ^(C) OE21 Head and Neck 3.89 3.89 3.89 ^(C) SCC-25 Head and Neck 5.99 5.99 5.95 ^(C) SCC-4 Head and Neck 8.51 8.51 8.47 ^(B) SCC-9 Head and Neck 7.35 7.42 7.39 ^(B) 769-P Kidney 1.05 1.05 1.05 ^(B) 786-O Kidney 2.81 2.81 2.81 ^(B) A498 Kidney 1.80 1.80 1.79 ^(B) A-704 Kidney 2.99 3.01 2.98 ^(B) ACHN Kidney 3.26 3.26 3.25 ^(A) Caki-1 Kidney 4.66 4.66 4.59 ^(C) Caki-2 Kidney 4.76 4.80 4.80 ^(A) G-401 Kidney 4.13 4.13 4.13 ^(B) SK-NEP-1 Kidney 3.99 3.99 3.99 ^(D) HepG2 Liver 1.54 1.54 1.54 ^(C) HLE Liver 3.92 3.92 3.89 ^(B) HLF Liver 3.99 3.99 3.96 ^(C) HuCCT1 Liver 3.64 3.64 3.64 ^(B) HUH-6 Clone Liver 1.70 1.70 1.69 ^(B) 5 OCUG-1 Liver 3.61 3.64 3.64 ^(B) SNU-423 Liver 1.47 1.47 1.45 ^(B) A427 Lung - NSCLC 4.13 4.13 4.10 ^(A) A549 Lung - NSCLC 2.98 2.98 2.98 ^(C) Calu1 Lung - NSCLC 2.72 2.72 2.69 ^(A) Calu6 Lung - NSCLC 9.56 9.77 9.59 ^(B) ChaGoK1 Lung - NSCLC 1.69 1.69 1.68 ^(A) COR-L105 Lung - NSCLC 2.85 2.86 2.84 ^(A) COR-L23 Lung - NSCLC 4.59 4.62 4.59 ^(D) Hs 229.T Lung - NSCLC 3.09 3.31 3.15 ^(A) NCI-H292 Lung - NSCLC 3.50 3.50 3.49 ^(A) NCIH441 Lung - NSCLC >10.5 >10.5 >10.5 ^(A) NCI-H460 Lung - NSCLC 2.99 2.99 2.99 ^(A) NCI-H520 Lung - NSCLC 3.02 3.02 3.01 ^(A) NCI-H596 Lung - NSCLC 8.89 8.93 8.86 ^(A) NCI-H661 Lung - NSCLC 1.70 1.70 1.69 ^(A) SKMES1 Lung - NSCLC 1.26 1.26 1.25 ^(A) DMS114 Lung - SCLC 4.62 4.69 4.55 ^(C) DMS53 Lung - SCLC 0.51 0.51 0.48 ^(A) NCIH446 Lung - SCLC 3.75 3.75 3.75 ^(B) NCI-H69 Lung - SCLC 5.01 5.01 4.73 ^(A) SHP-77 Lung - SCLC 1.03 1.03 1.03 ^(B) SW900 Lung - SCLC 3.64 3.68 3.61 ^(C) AsPC-1 Pancreas 2.51 2.51 2.45 ^(A) BxPC-3 Pancreas 4.48 4.48 4.48 ^(A) Capan-1 Pancreas >10.5 >10.5 >10.5 ^(C) Capan-2 Pancreas 1.34 1.34 1.33 ^(A) CFPAC4 Pancreas 3.57 3.57 3.57 ^(A) HPAF-II Pancreas 2.30 2.31 2.29 ^(C) Hs 766T Pancreas 2.44 2.47 2.42 ^(A) HuP-T4 Pancreas >10.5 >10.5 >10.5 ^(C) Mia PaCa-2 Pancreas 3.15 3.15 3.15 ^(A) PANC-1 Pancreas 2.20 2.20 2.17 ^(A) PSN-1 Pancreas 2.70 2.70 2.70 ^(B) SU.86.86 Pancreas >10.5 >10.5 >10.5 ^(C) YAPC Pancreas 3.37 3.37 3.34 ^(A) BeWo Placenta 10.05 >10.5 >10.5 ^(B) JAR Placenta 8.65 8.65 8.65 ^(C) JEG-3 Placenta 4.38 4.38 4.34 ^(B) 22Rv1 Prostate 4.13 4.13 4.10 ^(A) BM-1604 Prostate 3.89 3.89 3.85 ^(C) BPH1 Prostate 4.10 8.82 7.98 ^(A) DU145 Prostate 3.61 3.61 3.57 ^(A) LNCaP Prostate 3.17 3.17 2.62 ^(C) PC-3 Prostate 4.06 4.06 4.03 ^(A) A101D Skin 6.23 6.23 6.09 ^(B) (Melanoma) A375 Skin 1.41 1.41 1.41 ^(B) (Melanoma) A7 Skin 0.92 0.92 0.91 ^(B) (Melanoma) C32 Skin 1.71 1.71 1.36 ^(B) (Melanoma) CHL-1 Skin 1.59 1.59 1.59 ^(D) (Melanoma) COLO 829 Skin 1.01 1.03 0.98 ^(D) (Melanoma) G-361 Skin 2.70 2.70 2.69 ^(B) (Melanoma) HMCB Skin 1.46 1.47 1.46 ^(C) (Melanoma) Hs 294T Skin 1.73 1.73 1.67 ^(B) (Melanoma) Hs 688(A).T Skin 2.11 2.22 1.99 ^(B) (Melanoma) Hs 695T Skin 0.22 0.22 0.11 ^(C) (Melanoma) Hs 852.T Skin 3.64 3.64 3.50 ^(B) (Melanoma) Hs 934.T Skin 3.01 4.52 3.02 ^(B) (Melanoma) Hs 936.T(C1) Skin 2.26 2.26 2.22 ^(B) (Melanoma) MALME3M Skin 2.69 2.69 2.53 ^(C) (Melanoma) MeWo Skin 1.93 1.93 1.91 ^(B) (Melanoma) RPMI-7951 Skin 0.74 0.74 0.73 ^(C) (Melanoma) SH-4 Skin 2.60 2.60 2.58 ^(B) (Melanoma) SK-MEL-1 Skin 3.96 4.27 4.13 ^(B) (Melanoma) SK-MEL-28 Skin 2.38 2.39 2.37 ^(A) (Melanoma) SK-MEL-3 Skin 5.25 5.25 5.15 ^(C) (Melanoma) WM-266-4 Skin 1.19 1.19 1.18 ^(B) (Melanoma) G-292, clone Soft Tissue - 2.26 2.26 2.16 ^(B) A141B1 Osteosarcoma HOS Soft Tissue - 1.06 1.06 1.06 ^(B) Osteosarcoma Hs 888.Sk Soft Tissue - 3.09 3.19 3.10 ^(B) Osteosarcoma KHOS-240S Soft Tissue - 2.18 2.18 2.18 ^(D) Osteosarcoma MG-63 Soft Tissue - 9.66 9.70 9.66 ^(A) Osteosarcoma SaOS2 Soft Tissue - 3.36 3.43 3.33 ^(B) Osteosarcoma SJSA1 Soft Tissue - 1.51 1.51 1.50 ^(B) Osteosarcoma SW1353 Soft Tissue - 1.76 1.76 1.76 ^(B) Osteosarcoma U2OS Soft Tissue - 2.35 2.35 2.34 ^(C) Osteosarcoma A204 Soft Tissue - 0.65 0.65 0.64 ^(A) Sarcoma A-673 Soft Tissue - 1.15 1.15 1.15 ^(B) Sarcoma Hs 729 Soft Tissue - 3.07 3.11 3.01 ^(C) Sarcoma Hs 821.T Soft Tissue - 2.79 5.43 2.56 ^(B) Sarcoma HT-1080 Soft Tissue - 0.59 0.59 0.59 ^(A) Sarcoma MES-SA Soft Tissue - 1.53 1.53 1.53 ^(B) Sarcoma RD Soft Tissue - 0.69 0.69 0.69 ^(C) Sarcoma SJRH30 Soft Tissue - 1.46 1.46 1.46 ^(A) Sarcoma SK-LMS-1 Soft Tissue - 2.23 2.23 2.21 ^(C) Sarcoma SK-UT-1 Soft Tissue - 2.39 2.39 2.38 ^(B) Sarcoma SW684 Soft Tissue - 9.14 9.14 7.95 ^(C) Sarcoma SW872 Soft Tissue - 0.35 0.35 0.35 ^(A) Sarcoma SW982 Soft Tissue - 1.20 1.20 1.19 ^(B) Sarcoma TE 125.T Soft Tissue - 2.05 6.34 1.31 ^(B) Sarcoma TE 381.T Soft Tissue - 1.56 1.56 1.55 ^(A) Sarcoma VA-ES-BJ Soft Tissue - 4.17 4.17 4.13 ^(B) Sarcoma AGS Stomach 0.85 0.85 0.85 ^(B) HS 746T Stomach 3.99 6.51 4.94 ^(A) KATO III Stomach 4.62 4.62 4.62 ^(C) SK-PN-DW Stomach 2.16 2.16 2.15 ^(B) SNU-1 Stomach 1.51 1.51 1.51 ^(C) SNU-16 Stomach 1.44 1.44 1.44 ^(D) SNU-5 Stomach 4.80 4.80 4.73 ^(D) NTERA-2 Testis 1.98 1.99 1.96 ^(B) cl.D1 ^(A) 14 Jul. 2016; ^(B) 22 Aug. 2016; ^(C) 22 Sep. 2016; ^(D) 5 Oct. 2016

For Compound 1, a broad spectrum of activity was observed over the 280 cell types with very potent to strong (GI₅₀=0.006 to 2.98 μM) activity towards 174 cell lines. Very potent activity was observed towards SK-NEP-1 kidney (GI₅₀=0.00618 μM) and J-RT3-T3-5 leukemia (GI₅₀=0.0101 μM) cell lines. Moderately potent activity was observed (GI₅₀=0.11 μM) towards KG-1 leukemia cell line. Moderately potent activity (GI₅₀=0.4 to 1.0 μM) was observed towards the following cell lines: 2 breast cell lines KPL-1, MDA-MB-453; 3 myeloma cell lines IM-9, RPMI8226, SKO-007; 3 melanoma cell lines A7, COLO829, RPMI-7951; 7 soft tissue cell lines HOS (osteosarcoma), KHOS-240S (osteosarcoma), SW1353 (osteosarcoma), A204 (sarcoma), HT-1080 (sarcoma), RD (sarcoma), SW 872 (sarcoma); 2 small cell lung carcinoma cell lines DMS53, SHP-77; and also Colo 201 (colon); FaDu (head and neck), 769-P (kidney), HepG2 (liver), and AGS (stomach). Strong activity (GI₅₀=1 to 2.98 μM) was shown towards 149 cell lines: 16 melanoma; 15 lymphoma; 9 female genitourinary; 14 soft tissue (sarcoma); 16 colon; 12 leukemia; 8 non-small cell lung carcinoma; 8 pancreas; 9 breast; 10 CNS; 8 bladder; 5 kidney; 4 head and neck, 4 endocrine, 1 myeloma, 3 liver, 2 small cell lung carcinoma, 2 prostrate, 2 stomach and 1 testis.

For Compound 2, a narrow spectrum of activity was observed over the 280 cell types with potent to strong (GI₅₀=0.06 to 2.96 μM) activity towards 10 cell lines. Potent activity was observed towards SK-NEP-1 kidney (GI₅₀=0.0606 μM) Strong activity (GI₅₀=1 to 2.96 μM) was observed towards the following cell lines: 5 leukemia cell lines J-RT3-T3-5, Jurkat, MOLT-3, NALM-6, TF-1; 2 myeloma cell lines RPMI8226, SKO-007; 1 soft tissue cell line RD (sarcoma). For Compound 2 activities towards Jurkat, MOLT-3, NALM-6, TF-1 (leukemia) and SKO-007 (myeloma) were similar in magnitude to Compound 1.

For Compound 4, a narrow spectrum of activity was observed over the 280 cell types with moderately potent to strong (GI₅₀=0.68 to 2.92 μM) activity towards 12 cell lines. One strong activity (GI₅₀=0.684 μM) was observed towards A204 (soft tissue sarcoma) similar in magnitude to that observed for Compound 1. Strong activity (GI₅₀=1.23 to 2.92 μM) for Compound 4 was observed towards the following cell lines: 5 lymphoma (Daudi, DOHH-2, GA-10, L-428, ST486); 2 leukemia (MX1, NALM-6); also for T47D (breast), CEM-C1 (leukemia), A-673 (soft tissue sarcoma). The activity towards these cell lines is comparable in magnitude to Compound 1, however, Compound 4 show strong activity towards the J-RT3-T3-5 leukemia cell line (GI₅₀=2.71 μM) approximately 270 times less activity than the very potent activity observed for Compound 1 (GI₅₀=0.0101 μM) towards this cell line.

The plant resin extract showed strong activity (GI₅₀=0.11 to 1.03 μg/mL) towards 24 cell lines: 4 soft tissue sarcoma (A204, HT-1080, RD, SW 872); 4 melanoma (A7, COLO829, Hs695T, RPMI-7951); 3 myeloma (IM-9, RPMI8226, SKO-007); 2 breast (KPL-1, MDA-MB-453); 2 lymphoma (DOHH-2, SU-DHL-5); 2 small cell carcinoma (DMS53, SHP-77); 2 leukemia (Jurkat, NALM-6) and also bladder (UM-UC-3); colon (SW480); thyroid (CGTH-W-1); head and neck (FaDu); and stomach (AGS). For the Hs695T melanoma cell line, calculated on a weight-weight basis, the plant resin extract (GI₅₀=0.11 μg/mL) was approximately 3 times more active than Compound 1 (GI₅₀=0.37 μg/mL) towards this particular cell line. From weight comparison for all other cell lines the plant extract was less active than Compound 1.

Overall, Compound 1 provides a broad spectrum of anticancer activity towards a wide range of cell lines. Compound 2 shows specificity for the LNCaP prostrate cell line, also specific activity towards SK-NEP-1 (kidney), Jurkat, MOLT-3, NALM-6, TF-1 (leukemia) and SKO-007, RPMI8226 (myeloma) similar in magnitude to Compound 1. Compound 4 showed moderately potent to strong activity (GI₅₀=0.68 to 2.92 μM) towards a narrow range of cell lines (12 out of 280) compared with Compound 1 (174 out of 280),

TABLE 30 Number of cancer cell types for levels of growth inhibitory activity for Compounds 1, 2 and 4 Number of cell types GI₅₀ range Comp Comp Comp GI Activity (μM) 1 2 4 very potent 0.001-0.029   2 0 0 Potent 0.03-0.099  0 1 0 moderately potent 0.1-0.99  23 1 1 Strong 1-2.99 149 8 11 moderately strong 3-9.99 99 110 125 weak 10-30.00 6 118 130 very weak >30 1 42 13 TOTAL 280 280 280

TABLE 31 Number of cancer cell types for levels of growth inhibitory activity for M. insulare extract Number of cell types GI₅₀ range M. insulare GI Activity (μg/mL) extract very potent 0.00035-0.0104 0 Potent 0.0105-0.034 0 moderately potent 0.035-0.34 1 Strong  0.35-1.04 23 moderately strong  1.05-3.49 165 weak   3.5-10.49 83 very weak >10.5 8 TOTAL 280 Inhibition of Toll-Like Receptor 4 (TLR4) Activation on Human Peripheral Blood Mononuclear Cells (PBMC's). Study Objective

To evaluate compound capability to inhibit Toll-like receptor 4 (TLR4) activation on human peripheral blood mononuclear cells (PBMC's).

Experimental Protocol

Compound 1 was evaluated for ability to inhibit TLR4 in human PBMC's, as measured by release of specific cytokines. Evaluation was conducted by Eurofins Panlabs Inc., St Charles, Mo., USA.

TLR4 Inhibition Assay Protocol

-   -   a) Cryopreserved human PBMC's were drip-thawed.     -   b) Cells were diluted to the appropriate density (1×105 per         well) and seeded into 96-well polypropylene plates with 150 μL         per well of culture medium (RPMI 1640, 10% heat-inactivated FBS,         1% penicillin/streptomycin, 2 mM L-glutamine).     -   c) Cells were incubated at 37° C., 5% CO₂ for 1 hour prior to         the addition of test compounds or controls.     -   d) Test compounds were solubilized in DMSO to make stock         solutions, and then diluted further with cell culture medium.         The positive control, LPS (from S. minnesota R595), was         resuspended in endotoxin-free water at 0.5 mg/mL to make working         stock solutions. Dexamethasone was used as a reference control         compound.     -   e) Compounds and controls, including appropriate vehicle         controls, were added to the PBMC's in volumes of 10 μL and         incubated for 1 hour at 37° C., 5% CO₂. Compounds were tested in         duplicate at final assay concentrations of 10, 3, 1, 0.3, 0.1,         and 0.03 μM. Vehicle control wells simply received 10 μL of the         appropriate vehicle.     -   f) After a 1-hour incubation, 40 μL of diluted working stock LPS         were added to the test compound and control wells to give final         assay volumes of 200 μL. Plates were incubated for 24 hours at         37° C., 5% CO₂.     -   g) Plates were centrifuged at 200×g for 10 minutes. Cell culture         supernatants were collected and stored at −80° C. until needed         for analysis.     -   h) Cytokine levels in each sample were determined using Luminex         methodology, per the manufacturer's protocol.

PBMC Donor: Human donor 1 (lot #100)

Results and Discussion

Stimulation of human PBMC's with LPS for 24 hours elicited the measurable release of appropriate cytokines within expected ranges. Conversely, dexamethasone inhibited the LPS-stimulated release of cytokines from human PBMC's, also within expected ranges. Compound 1 elicited measurable inhibition of the LPS-induced release of specific cytokines from human PBMC's, following a 24-hour stimulation.

As seen from the results shown in Table 32, Compound 1 elicited a moderate to strong inhibition of the secretion of all cytokines at the top test concentration, giving measured IC₅₀ values of 4.3 μM, 6.6 μM, 8.8 μM, and 8.9 μM for IL-10, IL-1β, IL-6, and TNFα, respectively. (IC₅₀ values could not be measured for IL-8 and MIP-1α.)

TABLE 32 Compound 1 activity against certain LPS-induced cytokines Compound 1 LPS-induced cytokine Assay IC₅₀ (μM) I-1β 6.6 IL-6 8.8 IL-8 NA IL-10 4.3 MIP-1α NA TNF-α 8.9 Bioprint Assays

The Bioprint assay (conducted by Eurofins Cerep, France) provides a profile that is designed with the dual purpose of assessing safety at targets with known safety liabilities as well as providing a rich enough profile to search for compounds with similar profiles.

Methodology

Compound 1 was tested at 10 μM. For Compound 1 at 10 μM, the agonist radioligand assays for control specific inhibition of interest for biological activity or safety are as follows:—

GPCR Family:—adenosine A3 (h), 92%; adrenergic alpha 2C (h), 92%; cannabinoid CB2 (h), 71%; cholecystokinin CCK1 (CCKA) (h), 81%; dopamine D1 (h), 67%; dopamine D3 (h), 85%; histamine H1 (h), 64%; melatonin MT1 (ML1A) (h), 91%.

Transporters:—norepinephrine transporter (h), 76%.

Non-steroid Nuclear Receptors:—PPARgamma (h), 91%.

For Compound 1 at 10 μM, the enzyme assays for control specific inhibition of interest for biological activity or safety are as follows:—

AA Metabolism:—COX1 (h), 81%; COX2 (h), 74%.

Compound binding was calculated as a % inhibition of the binding of a radioactively labeled ligand specific for each target. Compound enzyme inhibition effect was calculated as a % inhibition of control enzyme activity. Results showing an inhibition or stimulation higher than 50% are considered to represent significant effects of the test compounds.

Results

Compound 1 results for inhibition of control specific binding to receptors are shown in Table 33 whilst results for inhibition of control specific binding inhibition of enzymes in enzyme and cell-based assays are shown in Table 34.

TABLE 33 Bioprint results for Compound 1 tested at 10 μM for inhibition of control specific binding to receptors. Compound 1 % Inhibition of Control Specific Reference Family Binding Assay Binding Compound IC₅₀ (μM) GPCR ADENOSINE A1 (h) (agonist 10 CPA 0.0021 radioligand) ADENOSINE A2A (h) (agonist 18 NECA 0.036 radioligand) ADENOSINE A2B (h) (antagonist 8 NECA 0.59 radioligand) ADENOSINE A3 (h) (agonist 92 IB-MECA 4.5E−04 radioligand) ADRENERGIC alpha 1A (h) 17 WB 4101 2.7E−04 (antagonist radioligand) ADRENERGIC alpha 1B (h) 41 prazosin 1.7E−04 (antagonist radioligand) ADRENERGIC alpha 2A (h) 35 yohimbine 0.0065 (antagonist radioligand) ADRENERGIC alpha 2B (h) 7 yohimbine 0.0065 (antagonist radioligand) ADRENERGIC alpha 2C (h) 92 yohimbine 0.004 (antagonist radioligand) ADRENERGIC beta 1 (h) (agonist 19 atenolol 0.34 radioligand) ADRENERGIC beta 2 (h) (agonist 2 ICI 118551 7.8E−04 radioligand) ADRENERGIC Adrenergic beta3 33 Alprenolol 0.25 ANGIOTENSIN II AT1 (h) (antagonist 60 saralasin 0.0019 radioligand) ANGIOTENSIN II AT2 (h) (agonist 54 angiotensin-II 1.4E−04 radioligand) APELIN APJ (apelin) (h) 24 apelin-13, TFA 3.4E−04 (agonist radioligand) BOMBESIN BB3 (h) (agonist 26 Bn(6-14) 0.0048 radioligand) BRADYKININ B2 (h) (agonist −37 NPC 567 0.038 radioligand) CANNABINOID CB1 (h) (agonist 46 CP 55940 0.001 radioligand) CANNABINOID CB2 (h) (agonist 71 WIN 55212-2 0.0022 radioligand) CYTOKINES TNF-alpha (h) −23 TNF-alpha 1.6E−04 (agonist radioligand) CHOLECYSTOKININ CCK1 (CCKA) (h) 81 CCK-8s 1.5E−04 (agonist radioligand) CHOLECYSTOKININ CCK2 (CCKB) (h) 6 CCK-8s 1.7E−04 (agonist radioligand) CORTICOTROPIN CRF1 (h) (agonist 34 sauvagine 2.5E−04 RELEASING radioligand) FACTOR DOPAMINE D1 (h) (antagonist 67 SCH 23390 3.9E−04 radioligand) DOPAMINE D2S (h) (agonist 59 7-OH-DPAT 0.0033 radioligand) DOPAMINE D3 (h) (antagonist 85 (+)butaclamol 0.0024 radioligand) ENDOTHELIN ETA(h) (agonist 6 endothelin-1 4.9E−05 radioligand) ENDOTHELIN ETB (h) (agonist 40 endothelin-3 1.7E−05 radioligand) GABA GABAB(1b) (h) −17 CGP 54626 0.0022 (antagonist radioligand) GLUCAGON glucagon (h) (agonist −20 glucagon 0.001 radioligand) CHEMOKINES CCR2 (h) (agonist 48 MCP-1 8.7E−05 radioligand) HISTAMINE H1 (h) (antagonist 64 pyrilamine 0.0023 radioligand) HISTAMINE H2 (h) (antagonist 37 cimetidine 0.66 radioligand) HISTAMINE H3 (h) (agonist 10 (R)alpha-Me-histamine 0.0016 radioligand) HISTAMINE H4 (h) (agonist 23 imetit 0.0047 radioligand) LEUKOTRIENES BLT1 (LTB4) (h) 27 LTB4 3.7E−04 (agonist radioligand) LEUKOTRIENES CysLT1 (LTD4) (h) 10 LTD4 6.3E−04 (agonist radioligand) MELANIN- MCH1 (h) (agonist −5 human MCH 7.8E−05 CONCENTRATING- radioligand) HORMONE MELANOCORTIN MC1 (agonist 11 NDP-alpha -MSH 1.2E−04 radioligand) MELANOCORTIN MC3 (h) (agonist 60 NDP-alpha -MSH 2.7E−04 radioligand) MELANOCORTIN MC4 (h) (agonist 37 NDP-alpha -MSH 4.0E−04 radioligand) MELATONIN MT1 (ML1A) (h) 91 melatonin 2.8E−04 (agonist radioligand) MOTOLIN motilin(h) (agonist −12 [Nleu13]-motilin 8.9E−04 radioligand) MUSCARINIC M1 (h) (antagonist 8 pirenzepine 0.031 radioligand) MUSCARINIC M2 (h) (antagonist 1 methoctramine 0.032 radioligand) MUSCARINIC M3 (h) (antagonist −3 4-DAMP 0.001 radioligand) MUSCARINIC M4 (h) (antagonist 19 4-DAMP 9.2E−04 radioligand) NEUROKININ NK1 (h) (agonist 62 [Sar9, Met(O2)11]-SP 3.7E−04 radioligand) NEUROKININ NK2 (h) (agonist 52 [Nleu10]-NKA (4-10) 0.0029 radioligand) NEUROPEPTIDE Y Y1 (h) (agonist 29 NPY 1.6E−04 radioligand) OPIOD & OPIOID- delta (DOP) (h) 62 DPDPE 0.002 LIKE (agonist radioligand) OPIOD & OPIOID- kappa (KOP) (agonist 39 U 50488 0.0012 LIKE radioligand) OPIOD & OPIOID- mu (MOP) (h) 43 DAMGO 5.3E−04 LIKE (agonist radioligand) OPIOD & OPIOID- NOP (ORL1) (h) −4 nociceptin 8.4E−04 LIKE (agonist radioligand) PLATELET PAF (h) (agonist 39 C16-PAF 0.0034 ACTIVATED radioligand) FACTOR PROSTANOID EP2 (h) (agonist 53 PGE2 0.0027 radioligand) PROSTANOID FP (h) (agonist 54 PGF2alpha 0.003 radioligand) PROSTANOID IP (PGI2) (h) (agonist 16 iloprost 0.017 radioligand) SEROTONIN 5-HT1A (h) (agonist −18 8-OH-DPAT 8.3E−04 radioligand) SEROTONIN 5-HT1B (antagonist 42 serotonin 0.013 radioligand) SEROTONIN 5-HT1D (agonist 18 serotonin 0.0026 radioligand) SEROTONIN 5-HT2A (h) (agonist 9 (±)DOI 4.7E−04 radioligand) SEROTONIN 5-HT2B (h) (agonist 70 (±)DOI 0.0067 radioligand) SEROTONIN 5-HT2C (h) (agonist 32 (±)DOI 2.4E−04 radioligand) SEROTONIN 5-HT4e (h) (antagonist 2 serotonin 0.19 radioligand) SEROTONIN 5-HT6 (h) (agonist 42 serotonin 0.18 radioligand) SEROTONIN 5-HT7 (h) (agonist 14 serotonin 4.4E−04 radioligand) SOMASTATIN sst1 (h) (agonist 18 somatostatin-28 5.5E−04 radioligand) SOMASTATIN sst4 (h) (agonist 40 somatostatin-14 0.0042 radioligand) UROTENSIN-II UT (h) (agonist 46 urotensin-II 9.5E−04 radioligand) VASOACTIVE VPAC1 (VIP1) (h) −9 VIP 2.7E−04 INTESTINAL (agonist radioligand) PEPTIDE VASOPRESSIN V1a (h) (agonist 12 [d(CH2)51, Tyr(Me)2]- 0.0015 radioligand) AVP VASOPRESSIN V2 (h) (agonist 11 AVP 0.0013 radioligand) TRANSPORTERS CHOLINE choline transporter −41 hemicholinium-3 0.0046 (CHT1) (h) (antagonist radioligand) DOPAMINE dopamine transporter (h) 63 BTCP 0.012 (antagonist radioligand) GABA GABA transporter −10 nipecotic acid 2.3 (antagonist radioligand) NOREPINEPHRINE norepinephrine 76 protriptyline 0.0039 transporter (h) (antagonist radioligand) SEROTONIN 5-HT transporter (h) 56 imipramine 0.0044 (antagonist radioligand) ION-CHANNELS GABA GABAA1 (h) (alpha −36 muscimol 0.053 1, beta 2, gamma 2) (agonist radioligand) GABA CHANNELS BZD (central) (agonist −31 diazepam 0.0094 radioligand) GABA CHANNELS Cl- channel (GABA- 42 picrotoxinin 0.33 gated) (antagonist radioligand) GLUTAMATE PCP (antagonist −11 MK 801 0.0096 CHANNELS radioligand) GLUTAMATE AMPA (agonist 4 L-glutamate 0.37 CHANNELS radioligand) GLUTAMATE kainate (agonist 15 kainic acid 0.023 CHANNELS radioligand) GLUTAMATE NMDA (antagonist 9 CGS 19755 0.35 CHANNELS radioligand) GLYCINE glycine (strychnine- 4 glycine 0.28 CHANNELS insensitive) (antagonist radioligand) NICOTINIC N neuronal alpha 4beta 2 −3 nicotine 0.0046 CHANNELS (h) (agonist radioligand) NICOTINIC N muscle-type (h) 1 alpha -bungarotoxin 0.0019 CHANNELS (antagonist radioligand) SEROTONIN 5-HT3 (h) (antagonist −2 MDL 72222 0.0086 CHANNELS radioligand) Ca²⁺ CHANNELS Ca2+ channel (L, 55 nitrendipine 1.3E−04 dihydropyridine site) (antagonist radioligand) Ca²⁺ CHANNELS Ca2+ channel (L, −30 diltiazem 0.057 diltiazem site) (benzothiazepines) (antagonist radioligand) Ca²⁺ CHANNELS Ca2+ channel (L, −4 D 600 0.027 verapamil site) (phenylalkylamine) (antagonist radioligand) Ca²⁺ CHANNELS Ca2+ channel (N) 5 omega -conotoxin 1.7E−06 (antagonist radioligand) GVIA K⁺ CHANNELS SKCa channel 14 apamin 9.7E−06 (antagonist radioligand) Na⁺ CHANNELS Na+ channel (site 2) 48 veratridine 5.9 (antagonist radioligand) NUCLEAR RECEPTORS NON-STEROID PPARgamma (h) 91 rosiglitazone 0.015 NUCLEAR (agonist radioligand) RECEPTORS STEROID AR (h) (agonist −9 mibolerone 0.0021 NUCLEAR radioligand) RECEPTORS STEROID Estrogen ER alpha (h) 40 Diethylstilbestrol 4.5E−04 NUCLEAR (agonist radioligand) RECEPTORS STEROID GR (h) (agonist 47 dexamethasone 0.0035 NUCLEAR radioligand) RECEPTORS OTHER RECEPTORS SIGMA sigma (non-selective) (h) 11 haloperidol 0.045 (agonist radioligand) THYROID Thyroid Hormone 9 Triiodothyronine 3.9E−05 HORMONE

TABLE 34 Bioprint results for Compound 1 tested at 10 μM for inhibition of control specific binding inhibition of enzymes in enzyme and cell-based assays. Compound 1 % Enzyme and Inhibition Reference Cell-based of Control IC₅₀ Family Assays Values Compound (μM) KINASES RTK FLT-1 kinase (h) 36 staurosporine 0.0098 (VEGFR1) RTK IRK (h) (InsR) −15 staurosporine 0.052 CTK Abl kinase (h) 14 staurosporine 0.11 CTK Fyn kinase (h) 6 PP1 0.11 CTK Lyn A kinase (h) 9 staurosporine 0.012 CTK ZAP70 kinase (h) 33 staurosporine 0.021 CMGC CDK2 (h) (cycA) 12 staurosporine 0.011 CMGC ERK2 (h) (P42mapk) 6 staurosporine 1 CMGC p38alpha kinase (h) 4 SB202190 0.033 CAMK CaMK2alpha (h) 2 AIP 0.092 OTHER NON-KINASE ENZYMES AA METABOLISM COX1(h) 81 Diclofenac 0.0078 AA METABOLISM COX2(h) 74 NS398 0.19 ATPASE ATPase (Na+/K+) 22 ouabain 0.25 MONOAMINE & acetylcholinesterase (h) 32 galanthamine 0.57 NEUROTRANSMITTER MONOAMINE & COMT (catechol- O-methyl −6 Ro 41-0960 0.061 NEUROTRANSMITTER transferase) MONOAMINE & MAO-A (antagonist 15 clorgyline 0.0012 NEUROTRANSMITTER radioligand) NO SYNTHASES inducible NOS −1 1400 W 0.029 PHOSPHODIESTERASES PDE2A1 (h) 43 EHNA 1.2 PHOSPHODIESTERASES PDE3B (h) −3 milrinone 1.2 PHOSPHODIESTERASES PDE4D2 (h) 55 Ro 20-1724 0.15 PHOSPHODIESTERASES PDE5 (h) (non-selective) 11 dipyridamole 0.84 PHOSPHODIESTERASES PDE6 (non-selective) 4 zaprinast 0.16 SERINE PROTEASES caspase-3 (h) −3 Ac-DEVD-CHO 0.0029 ASPARTIC PROTEASES BACE-1 (h) (beta - 0 OM 99-2 0.081 secretase) ASPARTIC PROTEASES HIV-1 protease 9 pepstatin A 2.4 METALLOPROTEASES ACE (h) 14 captopril 5.8E−04 METALLOPROTEASES ACE-2 (h) −3 AC-GG-26-NH2 0.2 METALLOPROTEASES MMP-1 (h) −2 GM6001 0.0054 METALLOPROTEASES MMP-2 (h) 0 GM6001 7.9E−04 METALLOPROTEASES MMP-9 (h) 1 GM6001 3.6E−04 MISCELLANEOUS MT3 (ML2) (agonist 31 melatonin 0.16 ENZYMES radioligand) MISCELLANEOUS xanthine oxidase/superoxide O2- scavenging −140 allopurinol 7.8 ENZYMES In Vitro Toxicity: Normal Cell Panel Toxicity Assay

The OncoPanel (Eurofins Panlabs Inc., St Charles, Mo., USA) normal cell proliferation assay measures the proliferation response of normal cells to drug treatments through high-content fluorescence imaging.

Methodology

Normal cells (see description in Table 35) were grown in special medium for each cell type. Cells were seeded into 384-well plates and incubated in a humidified atmosphere of 5% CO₂ at 37° C. Compounds (information in Table 36) were added the day following cell seeding. At the same time, a time zero untreated cell plate was generated. After a 3-day incubation period, cells were fixed and stained to allow fluorescence imaging of nuclei.

TABLE 35 Description of cells used in normal cell panel toxicity assay Normal cell name Cell type Source Catalog number CCD 841 CoN Colon epithelial ATCC CRL-1790 HMEC Mammary epithelial ATCC PCS-600-010 HREC Renal mixed epithelial ATCC PCS-400-012 Wi38 Lung fibroblast ATCC CCL-75

TABLE 36 Information for compounds used in normal cell panel toxicity assay Stock Amount conc. Stock volume Max final test Compound ID F.W. received (mg) (mM) (microL) conc. (microM) Carbonyl cyanide 3-chlorophenylhydrazone 204.62 5.70 10.00 2786 10.00 Compound 1 318.00 4.80 30.00 500 30.00 Cycloheximide 281.35 5.40 10.00 1919 10.00 Paclitaxel 853.91 N/A 0.30 1000 0.30 Staurosporine 466.50 1.00 1.00 2144 1.00

Compounds were serially diluted in half-log steps from the highest test concentration specified in the above table, and assayed over 10 concentrations with a maximum assay concentration of 0.1% DMSO.

The experimental procedure and calculations are similar to the Oncopanel 10-day incubation cell proliferation assay described previously.

Results

Compound 1 activity (EC₅₀ IC₅₀ and GI₅₀ (μM)) is shown against the reference compound paclitaxel in Table 37. Data on other reference compounds for comparison purposes is shown in Table 38.

TABLE 37 Compound 1 activity (EC₅₀ IC₅₀ and GI₅₀ (μM)) against paclitaxel Compound 1 Paclitaxel Cell Cell Cell Cell Cell Cell Count Count Count Count Count Count normal human EC₅₀ IC₅₀ GI₅₀ EC₅₀ IC₅₀ GI₅₀ Cell Line primary cells (μM) (μM) (μM) (μM) (μM) (μM) CCD 841 colon epithelial 4.86 14.3 5.27 0.0119 N/A 0.0221 CoN HMEC mammary 30 30 6.39 >30 >30 >30 epithelial HREC renal mixed 6.45 7.98 4.3 0.00172 0.0484 0.0016 epithelial Wi38 lung fibroblast 5.45 10.4 5.92 0.0039 0.00941 0.0036

TABLE 38 Further reference compound activity Carbonyl cyanide 3- Cycloheximide chlorophenylhydrazone Staurosporine Cell Cell Cell Cell Cell Cell Cell Cell Cell Count Count Count Count Count Count Count Count Count EC₅₀ IC₅₀ GI₅₀ EC₅₀ IC₅₀ GI₅₀ EC₅₀ IC₅₀ GI₅₀ Cell Line (μM) (μM) (μM) (μM) (μM) (μM) (μM) (μM) (μM) CCD 841 0.505 2.77 0.588 10 10 6.21 0.00024 0.00057 0.00019 CoN HMEC 3.65 10 0.306 >10 >10 0.175 0.00229 >1 0.00091 HREC 0.117 0.593 0.0369 0.553 1.52 0.308 0.00055 0.0027 0.0005 Wi38 0.285 0.871 0.369 8.84 8.84 6.15 0.00049 0.00074 0.00033

Table 39 shows representative data for the response when the cell line HREC is treated with Compound 1 in an OncoPanel Normal Cell Proliferation Assay.

TABLE 39 Compound 1 against HREC Relative cell count (%) Concentration (microM) Mean StdDev 9.55E−04 98.5 8.8 3.02E−03 94.0 7.4 9.53E−03 93.3 6.6 3.01E−02 96.0 8.3 9.52E−02 95.8 4.5 3.01E−01 96.7 8.7 9.51E−01 89.3 4.7 3.00E+00 85.1 14.4 9.49E+00 40.4 3.3 3.00E+01 20.9 5.7

The data displayed in Table 39 is graphed in FIG. 19.

DISCUSSION

Compound 1 and reference compounds, paclitaxel, cycloheximide, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and staurosporine, were tested on 4 normal cell types, CCD 841 CoN, HMEC, HREC and Wi38. In summary, Compound 1 showed relatively low toxicity towards all 4 cell lines. Paclitaxel showed low toxicity towards the HMEC cell line and high toxicity towards the other three cell lines. Based on average GI₅₀ for the 4 normal cell lines, the order of toxicity is staurosporine>paclitaxel>cycloheximide>CCCP>Compound 1.

In Vitro Toxicity: ADME-Toxicity Study

ADME-Toxicity study of compounds 1, 2 and 4 was conducted by Eurofins Panlabs Inc, St Charles, Mo., USA using a CYP panel.

Methodology

Cytochrome P450 Inhibition was monitored through HPLC-UV/VIS and HPLC-MS/MS detection. Peak areas corresponding to the metabolite of each substrate were recorded. The percent of control activity was then calculated by comparing the peak area obtained in the presence of the test compound to that obtained in the absence of the test compound. Subsequently, the percent inhibition was calculated by subtracting the percent control activity from 100 for each compound. IC₅₀ values (concentration causing a half-maximal inhibition of control values) were determined by non-linear regression analysis of the concentration-response curve using Hill equation curve fitting [Dierks, E. A. et al. (2001)].

Results

TABLE 40 Inhibition of CYPs by compounds 1, 2 and 4 against reference compounds % Inhibition of Control Values Human liver microsomes Compound 1 Compound 2 Compound 4 Reference In Vitro Metabolism 1 μM 10 μM 1 μM 10 μM 1 μM 10 μM Compound IC₅₀ μM CYP1A (phenacetin sub.) 28 92 38 63 14 85 Furafylline 16 CYP2B6 (bupropion sub.) 13 55 15 38 16 49 Clopidogrel 0.39 CYP2C8 (paclitaxel sub.) 14 76 12 52 24 61 Montelukast 0.57 CYP2C9 (diclofenac sub.) 5 77 20 72 31 91 Sulfaphenazole 0.28 CYP2C19 (omeprazole sub.) 37 90 34 73 25 78 Oxybutynin 6.7 CYP2D6 (dextromethorphan sub.) 13 45 18 44 21 45 Quinidine 0.12 CYP3A (midazolam sub.) 16 65 15 37 15 50 Ketoconazole 0.18 CYP3A (testosterone sub.) 7 77 17 57 20 48 Ketoconazole 0.18 Summary of Results Enzyme—Substrates CYP1A (Phenacetin Substrate)

Compounds 1, 2 and 4 are strong inhibitors at 10 μM (63-92%). With IC₅₀ values estimated to be <10, Compounds 1, 2 and 4 are stronger inhibitors the reference standard furafylline (IC₅₀16 μM). Compounds 1 and 2 showed moderate inhibition at 1 μM (28% and 38% respectively).

CYP2B6 (Bupropion Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (13-16% at 1 μM) compared with the reference compound clopidogrel (IC₅₀ 0.39 μM).

CYP2C8 (Paclitaxel Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (12-24% at 1 μM) compared with the reference compound montelukast (IC₅₀ 0.57 μM).

CYP2C9 (Diclofenac Substrate)

Compounds 1 was a very weak at 1 μM (5%) and Compounds 2 and 4 relatively weak inhibitors (20 and 31%) compared with the reference compound sulfaphenazole (IC₅₀ 0.28 μM).

CYP2C19 (Omeprazole Substrate)

Compounds 1, 2 and 4 are moderately strong inhibitors (25-37% at 1 μM) with activity comparable with oxybutynin (IC₅₀ 6.7 μM).

CYP2D6 (Dextromethorphan Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (12-21% at 1 μM) compared with the reference compound quinidine (IC₅₀ 0.12 μM).

CYP3A (Midazolam Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (15-16% at 1 μM) compared with the reference compound ketoconazole (IC₅₀ 0.18 μM).

CYP3A (Testosterone Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (7-20% at 1 μM) compared with the reference compound ketoconazole (IC₅₀ 0.18 μM).

In Vivo Toxicity: Compound 1 Maximum Tolerated Dose for Mice

Two studies were conducted by Eurofins Panlabs Taiwan, Ltd. The purpose of Study 1 was to assess the possible adverse effects of Compound 1 alone or in combination with carboplatin in Maximum Tolerated Dose (MTD) assay. The purpose of Study 2 is a follow-up study performed at escalating doses.

The vehicle information and dosing volume used in the studies are shown in Table 41. Table 42 and Table 43 show results for Study 1 in terms of mortality and body weight observations. In regard to Study 2, Table 44 and Table 45 show results for mortality and body weight observations and behavioral, symptomatic i.e. neurological and autonomic signs for Compound 1, alone and in combination with carboplatin, are shown in Table 46 and Table 47. Corresponding signs for the vehicles are shown in Table 48 and Table 49.

TABLE 41 Vehicle information and dosing volume for MTD assay studies Concen- Dosing Test tration volume Dosage Study article Vehicle (mg/mL) (mL/kg) (mg/kg) 1 Compound 1 10% Tween 20/ 10 10 100 90% PBS 2 Compound 1 20% Tween 20/ 20 10 200 80% PBS Test Substance and Dosing Pattern

Compound 1 was dissolved in 10% Tween 20/90% PBS or 20% Tween 20/80% PBS for IP injections. Compound 1 was administrated alone or in combination with carboplatin at a dosing volume of 10 or 20 mL/kg for each substance.

Study 1

For mortality, test substance (Compound 1; 100 mg/kg) alone or in combination with carboplatin (8.75, 17.5, or 35 mg/kg; given on the same day) was administered to mice by IP injection to evaluate the possible adverse effects in MTD assay. The mice were a group of 3 female NOD/SCID mice at 6-7 weeks of age. The mortality was observed at 30 min and again at 3, 24, 48 and 72 hours after compound administrations.

For body weight, test substance (Compound 1; 100 mg/kg) alone or in combination with carboplatin (8.75 or 17.5 mg/kg; given on the same day) was administered to a group of 3 female NOD/SCID mice at 6-7 weeks of age. The body weight of each animal was measured and recorded daily for 3 days.

Compound 1 alone did not elicit significant adverse effects and were considered tolerated. All test animals survived over the 72-hour observation period (Table 42). However, Compound 1 in combination with carboplatin was associated with slight to moderate limb tone, and resulted in a 33% mortality after 72 hours of dosing, reflecting the dose level was not well-tolerated (Table 42)

Compound 1, in combination with the lower doses of carboplatin (8.75 and 17.5 mg/kg), was further investigated in the MTD study. Compound 1, injected with two doses of carboplatin on the same day, was associated with slight to moderate abdominal tone during the first 30 minutes of administrations (Table 42 and Table 43). However, no mortality and body weight losses were observed in test animals over the experimental period, signifying the dose levels were tolerated (Table 42 and Table 43).

TABLE 42 MTD in mice (Study 1, Round 1 & 2) Dose Response (death/test) Compound Route (mg/kg) 30 min 3 hrs Day 1 Day 2 Day 3 Vehicle^(a) IP 10 mL/kg 0/3 0/3 0/3 0/3 0/3 Vehicle^(a) + IP 10 mL/kg + 0/3 0/3 0/3 0/3 0/3 Vehicle^(b) 10 mL/kg Compound 1 IP 100 mg/kg 0/3 0/3 0/3 0/3 0/3 Carboplatin IP 35 mg/kg 0/3 0/3 0/3 0/3 0/3 Compound 1 + IP 100 mg/kg + 0/3 0/3 0/3 0/3 1/3 carboplatin 35 mg/kg Vehicle^(a) + IP 10 mL/kg + 0/3 0/3 0/3 0/3 0/3 Vehicle^(b) 10 mL/kg Compound 1 + IP 100 mg/kg + 0/3 0/3 0/3 0/3 0/3 carboplatin 17.5 mg/kg Compound 1 + IP 100 mg/kg + 0/3 0/3 0/3 0/3 0/3 carboplatin 8.75 mg/kg Legend: ^(a)10% Tween 20/90% PBS ^(b)0.9% NaCl

TABLE 43 MTD in mice (Study 1; Round 2) Dose Body Weight (g) Compound Route (mg/kg) No. Day 0 Day 1 Day 2 Day 3 Vehicle^(a) + IP 10 mL/kg + 1 20 20 19 19 Vehicle^(b) 10 mL/kg 2 20 20 20 19 3 22 21 21 21 Compound 1 + IP 100 mg/kg + 1 22 21 21 20 carboplatin 17.5 mg/kg 2 22 22 22 21 3 21 21 21 21 Compound 1 + IP 100 mg/kg + 1 22 20 20 20 carboplatin 8.75 mg/kg 2 21 21 22 21 3 21 21 21 20 Legend: ^(a)10% Tween 20/90% PBS ^(b)0.9% NaCl Study 2

A follow-up MTD study was performed to evaluate for the adverse effects of the test substance (Compound 1) at escalating doses alone or in combination with carboplatin (17.5 mg/kg) via IP in a group of 3 female NOD/SCID mice at 6-7 weeks of age.

For mortality, treatment with Compound 1 at escalating doses (200 mg/kg) alone and in combination with carboplatin (17.5 mg/kg; given on the same day) were administered by IP injection to a group of 3 female NOD/SCID mice at 6-7 weeks of age. The mortality was monitored at 30 min and again at 3, 24, 48 and 72 hours after compound administrations.

For body weight, test substance (Compound 1) at escalating doses (200 mg/kg) alone or in combination with carboplatin (17.5 mg/kg; given on the same day) were administered by IP injection to a group of 3 female NOD/SCID mice at 6-7 weeks of age. The body weight of each animal was measured and recorded daily for 3 days.

On treatment with Compound 1 (200 mg/g) alone and in combination with carboplatin (17.5 mg/kg) exhibited a 33-100% mortality, but no marked weight loss during the study period (Table 44 and Table 45).

TABLE 44 MTD in mice (Study 2) Dose Response (death/test) Compound Route (mg/kg) 30 min 3 hrs Day 1 Day 2 Day 3 Vehicle^(a) IP 10 mL/kg 0/3 0/3 0/3 0/3 0/3 Vehicle^(a) + IP 10 mL/kg + 0/3 0/3 0/3 0/3 0/3 Vehicle^(b) 10 mL/kg PT# 1206425 (UOS-70) IP 200 mg/kg 0/3 0/3 1/3 3/3 3/3 (Compound 1) PT# 1206425 (UOS-70) IP 200 mg/kg + 0/3 0/3 0/3 1/3 1/3 (Compound 1) + 17.5 mg/kg carboplatin Vehicle^(a) IP 20 mL/kg 0/3 0/3 1/3 2/3 2/3 (20% Tween20/ 80% PBS) Legend: ^(a)20% Tween 20/80% PBS ^(b)0.9% NaCl

TABLE 45 MTD in mice (Study 2; Round 1 & 2) Dose Body Weight (g) Compound Route (mg/kg) No. Day 0 Day 1 Day 2 Day 3 Vehicle^(a) IP 10 mL/kg 1 23 22 23 23 (20% 2 24 23 23 23 Tween20/80% 3 23 22 22 22 PBS) Vehicle^(a) + IP 10 mL/kg + 1 22 21 20 19 Vehicle^(b) 10 mL/kg 2 22 22 21 19 3 22 20 20 20 Compound 1 IP 200 mg/kg 1 22 21 NA NA 2 21 NA NA NA 3 21 21 NA NA Compound 1 + IP 200 mg/kg + 1 21 20 20 21 carboplatin 17.5 mg/kg 2 22 22 NA NA 3 21 20 19 20 Vehicle^(a) IP 20 mL/kg 1 23 NA NA NA 2 22 22 NA NA 3 23 22 22 21 Legend: ^(a)20% Tween 20/80% PBS ^(b)0.9% NaCl

For behavioral, symptomatic i.e. neurological and autonomic signs, test substance (Compound 1; 200 mg/kg) alone or in combination with carboplatin (17.5 mg/kg; given on the same day) were administered by IP injection to a group of 3 female NOD/SCID mice at 6-7 weeks of age. The animals were then observed for presences of acute toxic symptoms and autonomic effects for 30 min after the first dose.

Pronounced behavioral effects such as decreased startle, touch response, pinna and placing were observed on Compound 1 treatment, alone and in combination with carboplatin (see Table 46). Decreased neurologic signs, such as spontaneous activity, righting, ataxia and low limb post were observed on Compound 1 treatment (Table 46). Hypothermia (decreased body temperature) was observed as an autonomic sign (Table 47)

TABLE 46 Behavioral and neurologic signs in mice for compound 1 (Study 2; Round 1) Treatment Compound 1 Compound 1 + carboplatin Route IP IP Dosage 200 mg/kg 200 mg/kg + 17.5 mg/kg No. 1 No. 2 No. 3 No. 1 No. 2 No. 3 BEHAVIORAL B.W. (g) 22 21 21 21 22 21 Irritability − − − − − − Hyperactivity − − − − − − Inc. Startle − − − − − − Inc. Touch − − − − − − Dec. Startle + + + + + + Response Dec. Touch Response + + + + + + Inc. Exploration − − − − − − Dec. Exploration + + + + + + Pinna + + + + + + Placing ± ± ± ± + ± NEUROLOGIC Tremor − − − + − + Dec. Spont. Activity + + + + + + Straub Tail − − − − − − Reactivity − + + + ± + Righting + + ± ± + + Ataxia + + ± ± + + Convulsion C.T.C-T − − − − − − Low Limb Post + + + + + + Abdominal Tone + + + + + + Limb Tone + + + + + + Grip Strength − ± + − + ±

TABLE 47 Autonomic signs in mice for compound 1 (Study 2; Round 1) Treatment Compound 1 Compound 1 + carboplatin Route IP IP Dosage 200 mg/kg 200 mg/kg + 17.5 mg/kg AUTONOMIC No. 1 No. 2 No. 3 No. 1 No. 2 No. 3 Skin Color − − − − − − Respiration D± D+ D+ D+ D+ D+ Salivation F.V. − − − − − − Lacrimation − − − − − − Diarrhea − − − − − − Body Temperature ↓+ ↓+ ↓+ ↓+ ↓+ ↓+ Piloerection − − − − − − Inc. Palpebral Size − − − − − − Dec. Palpebral Size − − − − ± − Others − − − − − − Death − − − − − −

TABLE 48 Behavioral and neurologic signs in mice for vehicles (Study 2; Round 1) Treatment Vehicle^(a) Vehicle^(a) + Vehicle^(b) Route IP IP Dosage 10 mL/kg 10 mL/kg + 10 mL/kg No. 1 No. 2 No. 3 No. 1 No. 2 No. 3 BEHAVIORAL B.W. (g) 23 24 23 22 22 22 Irritability − − − − − − Hyperactivity − − − − − − Inc. Startle − − − − − − Inc. Touch − − − − − − Dec. Startle − − − − − − Response Dec. Touch Response − − − − − − Inc. Exploration − − − − − − Dec. Exploration − − − − − − Pinna − − − − − − Placing − − − − − − NEUROLOGIC Tremor − − − − − − Dec. Spont. Activity − − − − − − Straub Tail − − − − − − Reactivity − − − − − − Righting − − − − − − Ataxia − − − − − − Convulsion C.T.C-T − − − − − − Low Limb Post − − − − − − Abdominal Tone − − ± ± ± ± Limb Tone − − − − − − Grip Strength − − − − − −

TABLE 49 Autonomic signs in mice for vehicles (Study 2; Round 1) Treatment Vehicle^(a) Vehicle^(a) + Vehicle^(b) Route IP IP Dosage 10 mL/kg 10 mL/kg + 10 mL/kg AUTONOMIC No. 1 No. 2 No. 3 No. 1 No. 2 No. 3 Skin Color − − − − − − Respiration − − − − − − Salivation F.V. − − − − − − Lacrimation − − − − − − Diarrhea − − − − − − Body Temperature − − − − − − Piloerection − − − − − − Inc. Palpebral Size − − − − − − Dec. Palpebral Size − − − − − − Others − − − − − − Death − − − − − − Legend for Tables 46 to 49 ^(a)20% Tween 20/80% PBS ^(b)0.9% NaCl −: no effects ±: Slight to moderate effects +: Severe effects Inc.: Increased Dec.: Decreased Spont.: Spontaneous C.: Chronic T.: Tonic C-T: Chronic-Tonic ↓: Low D: Deep In Vivo Anti-Cancer Activity: Compound 1 and Gemcitabine

An in vivo study indicates that Compound 1 is effective in combination with gemcitabine. The study design is detailed below and in Table 50. There is evidence for synergy from the study results which are shown in Table 51 and Table 52. Monotherapy and combinations were well tolerated.

TABLE 50 Tumor, Xenograft, Pancreas, MIA PaCa-2 in female nu/nu mice study design Test Conc. Dosage Mice^(a) Group Article Route mg/mL mL/kg mg/kg (female)  1^(#) Vehicle^(b) IP NA 10 NA 8 qwk x 3^(c) 2 Gemcitabine IP 8 10 80 8 q4d x 4^(d) 4 Compound 1 IP 10  10 100 8 qwk x 3^(c) 6 Compound 1 + IP + 10 + 8 10 100, qwk x 3^(c) + 8 Gemcitabine IP 80, q4d x 4^(d) ^(#)Negative control group ^(a)Female nu/nu mice aged 7-8 weeks are implanted with 1 × 10⁷ MIA PaCa-2 cells (0.2 mL/mouse), and dosing is initiated when group mean tumor volumes reach ~80-150 mm³. ^(b)Test substance vehicle (10% Tween20/90% Saline) ^(c)Doses are administered once weekly for 3 weeks. ^(d)Doses are administered once every four days (four total administrations). Body weight and tumor volumes are recorded twice weekly beginning on Day 1 and continuing until study completion. Tumor growth inhibition (% TGI) is determined twice weekly during the dosing period by the formula: % TGI = (1 − [(T − T0)/(C − C0)]) × 100 where T = mean tumor volume of treated group, T0 = mean tumor volume of treated group at study start, C = mean tumor volume of control group and C0 = mean tumor volume of control group at study start. Study is designed to continue for 28 days, or until the mean tumor volume within negative control group reaches 2000 mm³, whichever comes first. After TGI is determined, study may be converted to determine Tumor Growth Delay (TGD). Animals are to be sacrificed if/when: 1. Body weight loss >25% 2. Severe tumor ulceration Estimated Timeline

Animal Arrival: Within one month receiving test compounds

Tumor cells implantation: Within two weeks after animals are obtained

Dosing: Approximately 1-2 weeks after tumor cell implantation

Sample Requirements

Compound 1 150 mg; Gemcitabine 256 mg.

Protocol

Tumor, Xenograft Pancreas, MIA PaCa-2

Procedure: Groups of (8) female nu/nu mice (7-8 weeks old), bred in an animal isolator (IVC racks) under specific pathogens free (SPF) condition at 22 plus or minus 2° C. are used. Viable human pancreatic carcinoma MIA PaCa-2 (ATCC CRL-1420) cells (1.0×107 in 0.2 mL) are injected subcutaneously into the right flank of experimental mice. When tumor volumes reach ˜80-150 mm³ (about 8-10 days post implant), the animals are randomly assigned into groups of eight, and test compounds and/or vehicle dose administrations are initiated (denoted as Day 1). Test compounds are administered as detailed in the “Study Design” section (Table 50). Tumor volumes and body weights are measured and recorded twice weekly over the course of the study period. Animals are monitored as a group. The endpoint of the experiment is a mean tumor volume in the negative control group of 2000 mm³ or 28 days, whichever comes first.

Tumor volume (mm³) is estimated according to the formula for a prolate ellipsoid: length (mm)×[width (mm)]2×0.5. Tumor growth inhibition (% TGI) will be determined twice weekly during the dosing period by the formula: % TGI=(1−[(T−T0)/(C−C0)])×100 where T=mean tumor volume of treated group, T0=mean tumor volume of treated group at study start, C=mean tumor volume of control group and C0=mean tumor volume of control group at study start [Mohammed et al. 1998],

All aspects of this work including housing, experimentation, and animal disposal are performed in general accordance with the “Guide for the Care and Use of Laboratory Animals: Eighth Edition” (2011) in an AAALAC-accredited laboratory animal facility.

TABLE 51 Tumor volume change in a pancreas MIA PaCa-2 tumor xenograft model in response to treatment with compound 1 alone or in combination with Gemcitabine. Assay Tumor Xenograft, Pancreas, MIA PaCa-2 WO#1060729 (AB67928) Female nu/nu mice Dose (mg/kg) Tumor Volume (mm3) Gr. Treatment (Route) No. Day 1 Day 4 Day 8 Day 11 Day 15 1 Vehicle NA (mg/kg) 1 64 125 186 326 574 QWK x 3 2 74 65 87 128 151 IP 3 103 97 175 238 476 4 122 111 142 179 422 5 76 63 135 160 258 6 90 79 81 96 171 7 90 84 73 140 326 8 103 125 180 315 629 Mean 90 94 132 198 376 SEM 7 9 17 31 63 2 Gemcitabine 80 mg/kg 1 65 65 114 165 213 Q4D x 4 2 74 82 104 97 160 IP 3 104 128 151 215 419 4 115 123 162 228 335 5 76 73 75 103 130 6 88 100 70 85 128 7 91 58 70 94 115 8 101 119 113 127 156 Mean 89 94 107 139 207 SEM 6 10 13 20 39 % T/C 99 100 81 70 55 % TGI 1 0 19 30 45 4 Compound 1 100 mg/kg 1 67 56 126 368 600 QWK x 3 2 72 59 95 110 244 IP 3 104 105 125 192 276 4 115 103 107 121 197 5 81 129 187 266 414 6 87 70 111 188 309 7 93 92 177 251 409 8 100 107 138 188 312 Mean 90 90 133 211 345 SEM 6 9 12 30 45 % T/C 100 96 101 107 92 % TGI 0 4 −1 −7 8 6 Compound 1 + 100 mg/kg 1 69 46 48 57 80 Gemcitabine QWK x 3 2 70 111 94 104 116 IP + 3 108 86 101 97 103 80 mg/kg 4 111 132 124 157 265 Q4D x 4 5 84 68 71 35 95 IP 6 86 67 77 105 123 7 96 55 28 25 23 8 98 94 98 127 210 Mean 90 82 80 88 127 SEM 6 10 11 16 27 % T/C 100 87 61 44 34 % TGI 0 13 39 56 66

TABLE 52 Body weight change in a pancreas MIA PaCa-2 tumor xenograft model in response to treatment with Compound 1 alone or in combination with Gemcitabine. Assay # 581500 Tumor Xenograft, Pancreas, MIA PaCa-2 WO#1060729 (AB67928) Female nu/nu mice Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day 1 Day 4 Day 8 Day 11 Day 15 1 Vehicle NA (mg/kg) 1 28 27 27 27 26 QWK x 3 2 27 26 27 27 27 IP 3 25 24 25 26 27 4 24 24 24 24 24 5 26 25 25 25 25 6 25 25 25 26 26 7 25 23 23 24 24 8 25 25 24 25 26 Mean 25.6 24.9 25.0 25.5 25.6 SEM 0.5 0.4 0.5 0.4 0.4 2 Gemcitabine 80 mg/kg 1 24 23 25 25 26 Q4D x 4 2 30 28 30 31 31 IP 3 25 22 25 26 27 4 26 25 27 27 27 5 24 24 25 25 25 6 25 24 25 25 25 7 25 24 24 25 25 8 26 25 25 25 25 Mean 25.6 24.4 25.8 26.1 26.4 SEM 0.7 0.6 0.7 0.7 0.7 4 Compound 1 100 mg/kg 1 29 27 29 29 29 QWK x 3 2 26 24 26 25 25 IP 3 25 24 26 25 26 4 25 23 25 25 24 5 25 25 25 27 26 6 23 23 23 25 23 7 25 25 25 27 26 8 26 26 26 27 26 Mean 25.5 24.6 25.6 26.3 25.6 SEM 0.6 0.5 0.6 0.5 0.6 6 Compound 1 + 100 mg/kg 1 25 24 24 24 24 Gemcitabine QWK x 3 2 24 23 23 24 24 IP + 3 27 27 26 26 26 80 mg/kg 4 27 25 26 26 26 Q4D x 4 5 23 22 23 23 23 IP 6 26 25 25 25 24 7 27 26 26 26 26 8 25 24 24 23 23 Mean 25.5 24.5 24.6 24.6 24.5 SEM 0.5 0.6 0.5 0.5 0.5 Evaluation of Compound 1 to Treat Skin Diseases Aim

To evaluate compounds 1 with skin disease relevant in vitro assays. Evaluation was conducted by LEO Pharma Open Innovation, USA (openinnovation.leo-pharma.com).

Specific Objectives

-   -   1. Determination of inhibition of CCL2 release from IL-4, IL-13,         IL-22 and IFN-γ-stimulated primary human keratinocytes [assay         number 1280] and determination of the effect on cell viability         [assay number 1243],     -   2. Determination of inhibition of IL-8 release from IL-17A and         TNFα-induced primary human keratinocytes [assay number 1250] and         determination of the effect on cell viability [assay number         1244],     -   3. Determination of inhibition of IL-17A secretion from human         PBMC stimulated with antiCD3/antiCD28-coated beads [assay number         1321] and determination of the effect on cell viability [assay         number 1322],     -   4. Determination of inhibition of IL-4 and IL-2 secretion from         human CD4-positive T-cells stimulated with         antiCD2/antiCD3/antiCD28-coated beads [assay number 1297, 1298]         and determination of the effect on cell viability [assay number         1299],         Disease Relevance Summary     -   1. Compounds which inhibit keratinocyte CCL2 secretion may be         expected to have efficacy in atopic dermatitis.     -   2. Compounds which inhibit keratinocyte IL-8 secretion may be         expected to have efficacy in psoriasis.     -   3. Compounds which inhibit IL-17A secretion may be expected to         have efficacy in psoriasis.     -   4. Compounds which inhibit IL-4 secretion may be expected to         have efficacy in atopic dermatitis. Compounds which also inhibit         IL-2 secretion have a broader immunosuppressive effect since         IL-2 is an autocrine growth factor for T-cells.         Results     -   1. Effect of Compound 1 on monocyte chemoattractant protein-1         (MCP-1/CCL2) release in human keratinocytes induced by IL-4,         IL-13, IL-22 and IFN-γ.         -   FIG. 20A shows dose-responsive effect of Compound 1 on             chemokine CCL2 release in human keratinocytes induced by             IL-4, IL-13, IL-22 and IFN-γ.         -   Inhibition of CCL2 release: Rel EC₅₀=92.2 nM, Max             Effect=101%.         -   Inhibition of cell viability: Rel EC₅₀=708.0 nM, Max             Effect=103%.

Compound 1 showed medium potency in reduction of CCL2 release without significant reduction of cell viability as shown in FIG. 20A.

-   -   2. Effect of Compound 1 on IL-8 release in human keratinocytes         induced by IL-17 and TNF-α.         -   FIG. 20B shows dose-responsive effect of Compound 1 on IL-8             release in human keratinocytes induced by IL-17 and TNF-α.         -   Inhibition of IL-8 release: Rel EC₅₀=157 nM, Max             Effect=110%.         -   Inhibition of cell viability: Rel EC₅₀=246 nM, Max             Effect=101%.         -   There is similar reduction of IL-8 release and cell             viability.     -   3. Effect of Compound 1 on IL-17A release in Human PBMC         inflammation induced by CD3, CD28 and IL-23.         -   FIG. 20C shows dose-responsive effect of Compound 1 on             IL-17A release in Human PBMC inflammation induced by CD3,             CD28 and IL-23.         -   Inhibition of IL-17A release: Rel EC₅₀=320 nM, Max             Effect=100%.         -   Inhibition of cell viability: Rel EC₅₀=542 nM, Max             Effect=101%.         -   There is similar reduction of IL-17 release and cell             viability.     -   4. Effect of Compound 1 on IL-2 and IL-4 releases in T-cell         inflammation induced by CD2, CD3 and CD28.         -   FIG. 20D. Dose-responsive effect of Compound 1 on IL-2 and             IL-4 releases in T-cell inflammation induced by CD2, CD3 and             CD28.         -   Inhibition of IL-2 release: Rel EC₅₀=327 nM, Max Effect=98%.         -   Inhibition of IL-4 release: Rel EC₅₀=523 nM, Max             Effect=100%.         -   Inhibition of cell proliferation: Rel EC₅₀=587 nM, Max             Effect=123%.         -   There is similar effect on IL-2, IL-4 and cell             proliferation.             Discussion

Compound 1 showed pronounced effect in CCL2-release without significant effect on cell viability (FIG. 20A) compared to IL-8, IL-17A, IL-2 and IL-4 where the effect on release and cell viability are similar (FIGS. 20B to 20D). These results indicate that Compound 1 is a potential candidate for effective treatment of skin inflammatory diseases such as atopic dermatitis.

Methods

1. Keratinocyte CCL2 Release

Inhibition of CCL2 release from IL-4, IL-13, IL-22 and IFN-γ-stimulated primary human keratinocytes LEO Pharma Open Innovation assay number 1280

Disease Relevance

The inflammatory skin disease atopic dermatitis (AD) is characterized by the T-cell cytokines including IL-4, IL-13, IL-22 and IFN-γ. In the present assay, keratinocytes are stimulated with a mixture of these cytokines, and the release of CCL2 (C—C chemokine ligand 2, also called monocyte chemoattractant protein 1 (MCP-1)) is measured in the culture supernatant by proximity homogenous time-resolved fluorescence (HTRF). The purpose of the assay is to measure if test compounds inhibit the levels of CCL2 released by the keratinocytes. Compound which inhibit keratinocyte CCL2 secretion may be expected to have efficacy in atopic dermatitis.

A known steroid, Betamethasone, inhibits CCL2 release in this assay with an EC₅₀ of approximately 15 nM, and an Emax (plateau of the fitted curve) of approximately 60%. A high Emax shows that the compound inhibits a large proportion of the secreted CCL2. A low EC₅₀ value indicates that the compound is potent and to perform the inhibition at low concentrations.

Key assay parameters Description Human primary Cells are frozen in keratinocytes passage 2. Cells are (HEKa) used in passage 3-6 IL-4, IL-13, IL-22 and IFN-γ Atopic dermatitis-like stimulation: Recombinant human interleukin (IL) 4, 13 and 22 at 10 ng/mL and Interferon-γ at 1 ng/mL Effect Inhibition of AD stimuli induced CCL2 release from HEKa cells Positive control AD-like stimulation + Terfenadine (~100% effect) (CAS: 50679-08-8) at 10 microM Negative control AD-like stimulation (~0% effect) and 0.1% DMSO Reference compound Betamethasone (CAS: 378-44-9) Incubation time 48 h Capacity, run-time The assay takes 3 and requirements days to perform. Method Description CELL Culture:

HEKa are human epidermal keratinocytes isolated from adult skin. The cells have been cryopreserved at the end of the primary culture stage in a medium containing 10% DMSO. Sterile cell culture work applies.

Initiating Cultures from Cryopreserved HEKa Cells:

-   -   Thaw a vial of the frozen HEKa stock in a 37° C. water/bead         bath.     -   Transfer the cell suspension into 30 ml T75 cell culture flask.     -   Take 15 ml out and dispense over into 2×T75 (15 ml each).     -   Set up for assay 5-6 days later or pass it on.         Maintenance of Stock Cultures     -   Change the culture medium to freshly supplemented medium, 24 to         36 hours after establishing a secondary culture from         cryopreserved cells. For subsequent subcultures, change the         medium 48-72 hours after establishing the subculture.     -   Only passage up to 3-5 in the assay.         Subculture of HEKa     -   View and confirm 80% confluence.     -   Remove all of the culture medium from the flask.     -   Add 3 ml of TrypLE Express solution to the flask. Rock the flask         to ensure that the entire surface is covered.     -   Immediately remove all 3 ml of TrypLE Express solution from the         flask.     -   Add 1½ ml (T75) (3 ml T175) of fresh TrypLE Express solution to         the flask.     -   View the culture under a microscope. Incubate the flask at room         temperature until the cells have become completely round,         approximately 8-10 minutes.     -   Tap the flask very gently to dislodge cells from the surface of         the flask.     -   Add 3-4 ml (T75) (7 ml T175) of Trypsin Neutralizer solution to         the flask and transfer the detached cells to a sterile conical         tube.     -   Centrifuge the cells at 170×g for 7-10 minutes. Observe the cell         pellet.     -   Remove the supernatant from the tube, being careful not to         dislodge the cell pellet.     -   Resuspend the cell pellet in 15 ml medium with HKGS supplemented         (cat no S-001-K) but without hydrocortisone (when use for         assay). Pipette the cells up and down with a 10 ml pipette to         ensure a homogeneous cell suspension.     -   Determine the concentration of cells in the suspension.     -   Dilute the cells in supplemented medium and seed new culture in         a T175 flask. Incubate the cultures in a 37° C., 5% CO₂/95% air,         humidified cell culture incubator.         Assay Day 0:     -   Trypsinize the cells as described above.     -   Resuspend pellet in 10 ml assay medium (EpiLife Medium with HKGS         Kit but without hydrocortisone)     -   Filter the cells though a 37 μm strainer.     -   Count cells using a cell counter.     -   Seed HEKa cells in 384-well view plates 3500 c/w/40 μL.     -   Place the plates with lids on in a humidity chamber with lid         (24.5×24.5 cm box added H₂O in the bottom).     -   Incubate plates over night at 37° C., 5% CO₂/95% air         Assay Day 1:     -   Prepare compound-containing source plate, i.e. titrations in         DMSO to be transferred to the assay plate.     -   Add 80 nL compounds and controls from the source plate to the         assay plate (with cells).     -   Add 40 μL stimulation (recombinant human interleukin (IL) 4, 13,         22 at 10 ng/mL and Interferon-gamma at 1 ng/mL) in medium with         supplement, but without hydrocortisone.     -   Incubate plates at 37° C., 5% CO₂/95% air for 2 days.         Assay Day 3:     -   Remove plates from incubation and equilibrate to room         temperature for 30 minutes.     -   Transfer 8 μL supernatant to white proxi 384-well plates for         CCL2 detection.     -   Add 8 μL media to the detection plate.     -   Add 4 μL CCL2 HTRF detection reagent (to the white proxi 384w).     -   Seal and incubate for 4 hours and read in a plate reader.         Data Analysis and Calculations

CCL2 concentration in the supernatants is measured using homogeneous time-resolved fluorescence resonance (TR-FRET). The assay is quantified by measuring fluorescence at 665 nm (proportional to CCL2 concentration) and 620 nm (control). A ratio 665/620*1000 is calculated.

% Effect:

The capacity of the test compound to inhibit CCL2 release is normalized to the signal in the control wells with keratinocytes incubated with 0.1% DMSO (0%) and 10 μM Terfenadine (100%), which fully inhibits the CCL2 release.

2. Keratinocyte IL-8 Release

Inhibition of IL-8 release from IL-17A and TNFα-induced primary human keratinocytes LEO Pharma Open Innovation assay number 1250.

Disease Relevance

The inflammatory skin disease psoriasis is characterized by cytokines IL-17A and TNFα. In the present assay, primary human keratinocytes are stimulated with a mixture of these cytokines, and the release of IL-8 (also called CXCL8) is measured in the culture supernatant by proximity homogenous time-resolved fluorescence (HTRF). The purpose of the assay is to measure if the test compound is able to inhibit the levels of IL-8 released by the keratinocytes, indicating that it is able to inhibit part of the inflammation occurring in cells.

Compounds which inhibit keratinocyte IL-8 secretion may be expected to have efficacy in psoriasis.

A known steroid, Betamethasone, inhibits IL-8 release in this assay with an EC₅₀ of approximately 10 nM, and an Emax (plateau of the fitted curve) of approximately 50%. A high Emax indicates that the compound inhibits a large proportion of the IL-8 secretion. A low EC₅₀ value indicates that the compound is potent and performs the inhibition at low concentrations.

Key assay parameters Description Human epidermal Cells are frozen in keratinocytes, passage 2. Cells are adult (HEKa) used in passage 3-5 IL-17A and TNFα Psoriasis-like stimulation: Recombinant human interleukin (IL) 17A and tumor necrosis factor alpha (TNFα) both at 10 ng/mL Effect Inhibition of IL-17A/TNFα-induced IL-8 release from HEKa Positive control No stimulation (~100% effect) and 0.1% DMSO Negative control IL-17A/TNFα (~0% effect) stimulation and 0.1% DMSO Reference compound Betamethasone (CAS: 378-44-9) Incubation time 72 h Capacity, run-time The assay takes 3 and requirements days to perform. Method Description CELL Culture:

HEKa are human epidermal keratinocytes isolated from adult skin (See Cell Culture Method described for Keratinocyte CCL2 release above).

Assay Day 0:

-   -   Detach the cells as described above.     -   Resuspend pellet in 10 ml EpiLife Medium with HKGS Kit but         without hydrocortisone     -   Filter the cells though a 37 μm strainer.     -   Count cells using a cell counter.     -   Seed Human Epidermal Keratinocytes, adult (HEKa) cells in         384-well view plates 3500 c/w/40 μL. Use cell media with         supplement but without hydrocortisone as assay medium.     -   Place the plates with lids on in a humidity chamber with lid         (24.5×24.5 cm box added H₂O in the bottom).     -   Incubate plates over night at 37° C., 5% CO₂/95% air.         Assay Day 1:     -   Prepare compound-containing source plate, i.e. titrations in         DMSO to be transferred to the assay plate.     -   Empty the assay plate, and re-add 25 μL assay medium.     -   Add 75 nL compounds and controls from the source plate to the         assay plate (with cells).     -   Add 25 μL assay medium and incubate for 2 hours.     -   Add 25 μL stimulation mix in assay medium (recombinant human         interleukin 17A and Tumor necrosis factor-alpha) to give a final         concentration of 10 ng/mL of both cytokines.     -   Incubate plates at 37° C., 5% CO₂/95% air for 3 days.         Assay Day 4:     -   Remove plates from incubation and equilibrate to room         temperature for 30 minutes.     -   Transfer 2 μL supernatant to white proxi 384-well plates for         IL-8 detection.     -   Add 2 μL assay medium     -   Prepare IL-8 HTRF mix (according to manufactory protocol) and         add 2 μL pr well.     -   Seal and incubate for 3 hours and read fluorescence in a plate         reader.         Data Analysis and Calculations

IL-8 concentration in the supernatant is measured using homogeneous time-resolved fluorescence resonance (TR-FRET). The assay is quantified by measuring fluorescence at 665 nm (proportional to IL8 concentration) and 620 nm (control). A ratio 665/620*1000 is calculated as a representation of the amount of IL-8 in the supernatant.

% Effect:

The capacity of the test compound to inhibit IL-8 release is normalized to the signal in the negative control wells with keratinocytes incubated with 0.1% DMSO (0%) and 10 μM Terfenadine (100%), which fully inhibits the release of IL-8.

3. PBMC IL-17 Assay

Inhibition of IL-17A secretion from human PBMC stimulated with antiCD3/antiCD28-coated beads. LEO Pharma Open Innovation assay number 1321

Disease Relevance

The inflammatory skin disease psoriasis is characterized by cytokines IL-17A and TNFα. In the present assay, human peripheral blood mononuclear cells (PBMC) are stimulated with beads coated with antibodies against CD3 and CD28 to activate the T-cell receptor, and stimulated with IL-23 to promote T-helper17 activity. The cells are incubated for three days, then the secreted level of IL-17A is measured in the culture supernatant using AlphaLISA and the amount of living cells is measured by addition of resazurin (PrestoBlue®) to identify compounds that inhibit cell proliferation or viability. The purpose of the assay is to measure if test compounds inhibit the secretion of IL-17A by the PBMC. Compounds which inhibit IL-17A secretion may be expected to have efficacy in psoriasis.

The reference compound in this assay is the calcineurin inhibitor Tacrolimus and inhibits IL-17A release with an EC 50 of approximately 0.34 nM, and with an Emax (plateau of the fitted curve) of approximately 90%. A high Emax shows that the compound inhibits a large proportion of the secreted IL-17. A low EC₅₀ value indicates that the compound is potent and performs the inhibition at low concentrations.

Key Assay Parameters

Parameter Description Cells Human PBMC isolated from huffy coats and kept at −150° C. until day of assay Stimulation Beads coated with antibodies to CD3 and CD28 (T-cell activation/expansion kit Miltenyi Biotec cat no 130-091-441) One bead/cell. Effect Inhibition of IL-17A release 0% effect of Bead stimulation, test compound 0.1% DMSO 100% effect of Bead stimulation, 0.1% DMSO, test compound Terfenadine 10 uM Reference compound Tacrolimus (Cas no 104987-11-3-P) Incubation time 3 days Capacity, run-time The assay takes 3 days to perform. and requirements Method Description PBMC Isolation:

-   -   PBMC are isolated from fresh human huffy coats by centrifugation         on Lymphoprep® following instructions from Sebmate®     -   Cells are spun down and resuspended in freezing medium         (RPMI1640+20% FBS+5% DMSO)     -   Cells are frozen in aliquots of 8×10⁷ cells/vial matching one         384 plate. Cells are placed in Coolcell box and placed in         −80° C. freezer for one day. Vials are moved to the −150° C.         freezer in a prechilled box.         Assay Day 1:     -   Prepare assay medium: RPMI1640+10% heat inactivated FBS and 1%         pen/strep, supplemented with human IL-23, 20 ng/mL (R&D systems,         cat no 1290-IL-010)     -   Test compounds: Prepare compound-containing source plate, i.e.         titrations in DMSO to be transferred to the assay plate.     -   Add 70 nL compounds and controls from the source plate to the         assay plate.     -   Thaw the cells by adding 1 vial to 20 ml warm assay medium     -   Centrifuge the cells down at 170 G in 10 minutes, remove the         supernatant and resuspend in new medium.     -   Filter the cells though a 37 μm strainer.     -   Count the cells in the cell counter.     -   Beads (T Cell Activation/Expansion Kit-human, Miltenyi Biotech,         cat. no. 130-091-441): Coat beads with antibodies (antiCD3 and         antiCD28) according to the kit procedure.     -   Add the beads to the cells, one bead per cell, 2.1e+06 cells/ml.         Use a pipette to mix the cells and beads carefully.     -   Seed the cell/bead mixture into the assay plate, 70 μL/well         (1.3e+05 cells/well)     -   Place the plates in a humidity chamber with lid (24.5×24.5 cm         box added H₂O in the bottom).     -   Incubate plates at 37° C., 5% CO₂/95% air for 3 days.         Assay Day 4:     -   IL-17A concentration in the supernatants is measured using an         alphaLISA kit (Perkin Elmer, cat no. AL219F).     -   Pretest to find optima dilution of samples: Test 5 μL         supernatant from control wells and run IL-17A alphaLISA assay         with only 2 hours of incubation.     -   When data are available, remove assay plates from incubation and         equilibrate to room temperature for 30 minutes.     -   Transfer 5 μL or corrected amounts of supernatant to a 384-proxy         plate     -   Add 5 μL alphaLISA acceptor beads.     -   Incubate 1 hour at room temperature     -   Add 5 μL donor beads     -   Incubate overnight at room temperature protected from light.     -   Add 7 μL PrestoBlue reagent (resazurin)/well to the assay plate         and incubated for 2-4 hours at 37° C./5% CO₂. The fluorescence         in the wells is measured (Ex615/Em 535).         Assay Day 5:     -   Read alphaLISA plate in a plate reader         Data Analysis and Calculations

IL-17A concentration in the supernatants is measured using an alphaLISA kit (Perkin Elmer, cat no. AL219F).

% Effect IL-17:

The capacity of the test compound to inhibit IL-17A release is normalized to the signal in the negative control wells treated with atoxic compound, Terfenadine.

The level of living cells in the wells is measured by the addition of PrestoBlue® and the conversion of Resazurin to Resorufin (fluorescent) in living cells is measured.

% Effect Viability/Proliferation:

The effect of the test compounds on viability/proliferation is normalized to the PrestoBlue signal in the negative control wells treated with a toxic compound, Terfenadine.

4 and 5. T-Cell IL-2 & IL-4 Assay

Inhibition of IL-4 and IL-2 secretion from human CD4-positive T-cells stimulated with antiCD2/antiCD3/antiCD28-coated beads. LEO Pharma Open Innovation assay number 1297, 1298, 1299

Disease Relevance

The inflammatory skin disease atopic dermatitis is characterized by the T-cell cytokines IL-4, IL-13 and IL-22. In the present assay, human T-cells are stimulated with beads coated with antibodies against CD2, CD3 and CD28 to activate the T-cell receptor. Then the level of secreted of IL-4 is measured in the culture supernatant using electrochemiluminescence (MSD kits, Meso Scale Discovery), IL-2 is measured by proximity homogenous time-resolved fluorescence (HTRF) and the amount of living cells is measured by addition of resazurin (PrestoBlue®) to allow identifying compounds that inhibit T-cell proliferation or viability. The purpose of the assay is to measure if a test compound is able to inhibit the secretion of IL-4 by the T-cells, indicating that it is able to inhibit part of the inflammation occurring in the skin. Compounds which inhibit IL-4 secretion may be expected to have efficacy in atopic dermatitis. Compounds which also inhibit IL-2 secretion have a broader immunosuppressive effect since IL-2 is an autocrine growth factor for T-cells. A known steroid, Betamethasone, inhibits IL-4 release in this assay with an EC₅₀ of approximately 15 nM, and with an Emax (plateau of the fitted curve) of approximately 80%. A high Emax shows that the compound is able to inhibit a large proportion of the secreted IL-4. A low EC 50 value indicates that the compound is potent and is able to perform the inhibition at low concentrations.

Key Assay Parameters

Human CD4-postive T-cells isolated from buffy coats and kept at −150 degree C. until day of assay Stimulation Beads coated with antibodies to CD2, CD3 and CD28 (T-cell activation/expansion kit Miltenyi Biotec cat no 130-091- 441) One bead/cell. Effect Inhibition of IL-4 release from T-cells 0% effect of test compound Bead stimulation, 0.1% DMSO 100% effect of test compound Unstimulated cells, 0.1% DMSO Reference compound Betamethasone (CAS: 378-44-9) Incubation time 48 h Capacity, run-time The assay takes 3 and requirements days to perform. Method Description CD4+ T-Cell Isolation:

-   -   Peripheral blood mononuclear cells (PBMC) are isolated from         fresh human huffy coats by centrifugation on Lymphoprep®         following instructions from Sebmate™ (Stemcell).     -   The cells are resuspended in 30 ml of PBS+2% Serum and counted.     -   CD4 positive T-cells are isolated from freshly isolated PBMC         using EasySep™ humane CD4+ T-cell Isolation Kit (Catalog #17952)         and following the manufacturer's protocol.     -   Cells are spun down and resuspended in freezing medium (X-vivo         15+10% FBS+5% DMSO)     -   Cells are frozen in aliquots of 3×10⁷ cells/vial matching one         384 plate. Cells are place in Coolcell box and place in −80° C.         freezer for one day then transferred to the −150° C. freezer in         a prechilled box.         Assay Day 1:     -   Prepare assay medium: 500 ml X-Vivo 15+5 ml Glutamax (2 mM         final)+5 ml pen/strep (100 U/ml final).     -   Add 5 μL medium to the wells of a 384 well plate (Greiner cat.         No 788986).     -   CD4+ Cells: Thaw frozen cells from −150° C. freezer in 37° C.         bath, and pipette the cells into 10 ml medium. Spin 10 min @ 170         g.     -   Resuspend pellet in 3.5 ml per 2.5×10⁷ cells and add 10 μL per         well to the plate (40000 cells/well)     -   Test compounds: Prepare compound-containing source plate, i.e.         titrations in DMSO to be transferred to the assay plate.     -   Add 25 nL compounds and controls from the source plate to the         assay plate (with cells).     -   Beads (T Cell Activation/Expansion Kit-human, Miltenyi Biotech,         cat. no. 130-091-441): Coat beads with antibodies according to         the kit procedure.     -   Add 10 μL beads to each well except for the negative controls.         One bead per cell. Add 10 μL medium to the negative controls.     -   Incubate plates at 37° C., 5% CO₂/95% air for 2 days.         Assay Day 3:     -   Remove plates from incubation and equilibrate to room         temperature for 30 minutes.     -   IL-4 concentration in 10 μL supernatant is measured using an MSD         IL-4 384-plate kit (Custom made) according to manufacturer         protocol.     -   IL-2 concentrations in the supernatant is measured by an IL-2         HTRF kit, CisBio, cat. No 62HIL02PEH.     -   Add 4 μL medium to white proxi 384-well plates     -   Add 1 μL supernatant.     -   Add 4 μL mHTRF mix (each diluted 1:300 in ⅔ reconstitution         buffer and ⅓ assay medium).     -   Spin the plates for 1 minute at 170 g     -   Seal and incubate overnight and read fluorescence in a plate         reader     -   The amount of viable cells is measured by PrestoBlue® reagent     -   Add 15 μL diluted PrestoBlue® reagent (resazurin) corresponding         to 2.5 μL Prestoblue/well to the assay plate.     -   Incubate overnight at 37° C./5% CO₂. The fluorescence in the         wells is measured (Ex615/Em 535).         Data Analysis and Calculations

IL-4 concentration in the supernatants is measured using electrochemiluminescence (MSD kits, Meso Scale Discovery). IL-2 concentration in the supernatants is measured using homogeneous time-resolved fluorescence resonance (TR-FRET). The assay is quantified by measuring fluorescence at 665 nm (proportional to IL-2 concentration) and 620 nm (control). A ratio 665/620*1000 is calculated.

% Effect for IL-2 and IL-4:

The capacity of the test compound to inhibit IL-4 and IL-2 release is normalized to the signal in the negative control wells with un-stimulated T-cells.

The level of living cells in the wells is measured by the addition of PrestoBlue® and the conversion of Resazurin to Resorufin (fluorescent) in living cells is measured.

% Effect for Viability/Proliferation:

The effect of the test compounds on viability/proliferation is normalized to the PrestoBlue signal in the negative control wells treated with a toxic compound, Terfenadine.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

REFERENCES

-   Ghisalberti E L (1994) The phytochemistry of the Myoporaceae.     Phytochemistry, 35: 7-33. -   Davis R A, Caroll A R, Pierens G K (1999). New lamellarin alkaloids     from the Australian ascidian, Didemnum chartaceum. J. Nat. Prod,     62(3), 419-24. -   Ullman E F, Kirakossian H, Singh S, Wu Z P, Irvin B R, Pease J S,     Switchenko A C, Irvine J D, Daffom A, Skold C N (1994) Luminescent     oxygen channeling immunoassay: Measurement of particle binding     kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA Vol. 91,     5426-5430. -   Ma H, Deacon S, Horiuchi K (2008). The challenge of selecting     protein kinase assays for lead discovery optimization. Expert Opin     Drug Discov. 3(6): 607-621. -   Filippakopoulos, P., Picaud S, Mangos M, Keates T, Lambert J P,     Barsyte-Lovejoy D, Felletar I, Volkmer R, Müller S, Pawson T,     Gingras A C, Arrowsmith C H, Knapp S. (2012) Histone recognition and     large-scale structural analysis of the human bromodomain family.     Cell, 149(1): 214-31. -   Filippakopoulos, P. and S. Knapp (2012) The bromodomain interaction     module. FEBS Lett, 586(17): 2692-704. -   Philpott, M., Yang J, Tumber T, Fedorov O, Uttarkar S,     Filippakopoulos P, Picaud S, Keates T, Felletar I, Ciulli A, Knapp     S, Heightman T D (2011) Bromodomain-peptide displacement assays for     interactome mapping and inhibitor discovery. Mol Biosyst, 7(10):     2899-908. -   Kaustov, L., Ouyang H, Amaya M, Lemak A, Nady N, Duan S, Wasney G A,     Li Z, Vedadi M, Schapira M, Min J, Arrowsmith C H (2011) Recognition     and specificity determinants of the human cbx chromodomains. J Biol     Chem 286(1): 521-9. -   Kim, J., Daniel J, Espejo A, Lake A, Krishna M, Xia L, Zhang Y,     Bedford M T (2006) Tudor, MBT and chromo domains gauge the degree of     lysine methylation. EMBO Rep, 7(4): 397-403. -   Org, T., Chignola F, Hetenyi C, Gaetani M, Rebane A, Liiv I, Maran     U, Mollica L, Bottomley M J, Musco G, Peterson P (2008) The     autoimmune regulator PHD finger binds to non-methylated histone H3K4     to activate gene expression. EMBO Rep, 9(4): 370-6. -   Venturing L., You J, Stadler M, Galien R, Lallemand V, Koken M H,     Mattei M G, Ganser A, Chambon P, Losson R, de Thé H. (1999) TIF1     gamma, a novel member of the transcriptional intermediary factor 1     family. Oncogene, 18(5): 1209-17. -   Xie, S., J. Jakoncic, and C. Qian (2012) UHRF1 double tudor domain     and the adjacent PHD finger act together to recognize     K9me3-containing histone H3 tail. J Mol Biol, 415(2): 318-28. -   Adams-Cioaba, M. A., Li Z, Tempel W, Guo Y, Bian C, Li Y, Lam R,     Min J. (2012) Crystal structures of the Tudor domains of human PHF20     reveal novel structural variations on the Royal Family of proteins.     FEBS Lett, 586(6): 859-65. -   Hayashi-Takanaka Y, YamagataK, Wakayama T, Stasevich T J, KainumaT,     Tsurimoto T, Tachibana M, Shinkai Y, Kurumizaka H, Nozaki N, Kimura     H (2011) Nucleic Acids Res. 39(15): 6475-88. -   Kubicek, S. et al. (2007), Mol. Cell. 25:473-481. -   Ken-ichi Noma and Shiv I. S. Grewal (2002), Proc Natl Acad Sci USA.     Dec. 10, 1999 (Suppl 4): 16438-16445. -   Rotili, D. and Mai, A. (2011), Genes & Cancer, 2: 663-679. -   Chowdhury, R. et al. (2011), Eur. Mol. Biol. Org., 12: 463-469. -   Nottke, A. et al. (2009), Development, 136:879-889. -   Kristensen, L. H. et al. (2012), FEBS Journal, 279:1905-1914. -   Heightman T. D. (2011), Current Chemical Genomics, 5: 62-71. -   King O. N. F. et al. (2010), PLoS ONE, 5:1-12. -   Hong, S. et al. (2007), PNAS, 104:18439-18444. -   Pradhan, S. et al. (1999), J. Biol. Chem., 274: 33002-33010. -   Suetake I, Shinozaki F, Miyagawa J, Takeshima H, Tajima S. (2004), J     Biol Chem. 279(26): 27816-23. -   Aoki, A. et al. (2001), Nucleic. Acids. Res., 29: 3506-3512. -   Zhang, H. et al. (2012), J Biol Chem., 287(9):6573-81. -   Strahl, B. D. and C. D Allis (2000) The language of covalent histone     modifications. Nature, 403(6765): 41-5. -   Michan, S. and Sinclair, D. (2007), Biochem. J., 404: 1-13. -   Michishita, E. et al. (2008), Nature, 452, 492-496. -   Kim, W. and Kim, J. E. (2013), J. Physiol. Pharmacol.,     64(5):531-534. -   An S I, Yeo K J, Jeon Y H, Song J J. (2011), J Biol Chem. 286(10):     8369-74. -   Yost, J. M. et al. (2011), Curr. Chem. Genomics, 5: 72-84. -   Shen, X, Liu, Y. et al. (2008), Mol. Cell, 32: 491-502 -   Nayak, V. et al. (2011), Nucleus, 1:2. -   Jiang H I, Lu X, Shimada M, Dou Y, Tang Z, Roeder R G. (2013), Nat     Struct Mol Biol., 10:1156-63. -   Ali M1, Horn R A1, Blakeslee W1, Ikenouye L1, Kutateladze T G2.     (2014), Biochim Biophys Acta.; 1843(2):366-71. -   Allali-Hassani A1, Kuznetsova E1, Hajian T1, Wu H1, Dombrovski L1,     Li Y1, Graslund S1, Arrowsmith C H2, Schapira M3, Vedadi M4. (2014),     J Biomol Screen.; 19 (6):928-935. -   Selvi B. R. et al. (2010), Biochim Biophys Acta., 1799(10-12):     810-28. -   Munoz-Fuentes, V. et al. (2011), PLoS ONE, 6: 1-7. -   Cheng, D. et al. (2004), J. Biol. Chem., 279: 23892-23899. -   Li, K. K. et al. (2012), Med Res Rev. 32(4):815-67. -   Selvi, B. R. et al. (2010), J. Biol. Chem., 285: 7143-7152. -   Yost, J. M. et al. (2011), Curr. Chem. Genomics, 5: 72-84. -   Iberg, A. N. et al. (2007), J Biol. Chem., 283: 3006-3010. -   Zurita-Lopez C I1, Sandberg T, Kelly R, Clarke S G. (2012), J Biol     Chem. 287(11):7859-70. -   Lee, J. et al. (2005), The journal ofbiological chemistry, vol. 280,     38: 32890-32896. -   Du, H.-N. et al. (2008), Gene Dev, 22: 2786-2798 -   Schultz, D. C. et al. (2002), Genes Dev., 16: 919-932. -   Kang, H. B. et al. (2009), FEBS LETT 583: 1880-1886. -   Sabbattini P, Canzonetta C, Sjoberg M, Nikic S, Georgiou A,     Kemball-Cook G, Auner H W, Dillon N. (2007), EMBO J. 26(22):4657-69. -   Preuss U, Landsberg G, Scheidtmann K H. (2003), Nucleic Acids Res.,     31(3):878-85. -   Han A, Lee K H, Hyun S, Lee N J, Lee S J, Hwang H, Yu J. (2011),     Bioorg Med Chem., 19(7):2373-7. -   Baek S H. (2011), Mol Cell, 42(3):274-84 -   Barnett, S. F. et al. (2005), Biochem. J., 385: 399-408. -   Misaghi, S. et al. (2009), Mol. Cell. Biol. 29: 2181-2192. -   Horton, R. A. et al. (2007), Anal. Biochem., 360:138-143. -   Hu, M. et al. (2005), The EMBO Journal 24, 3747-3756. -   Ye Y. et al. (2011), EMBO reports, 12: 350-357. -   Tian, X. et al. (2011), Assay and Drug Dev. Technol., 9:165-173. -   Arrowsmith C H, Bountra C, Fish P V, Lee K, Schapira M (2012)     Epigenetic protein families: a new frontier for drug discovery.     Nature Reviews Drug Discovery, 11, 384-400. -   Plass C, Pfister S M, Lindroth A M, Bogatyrova O, Claus R and     Lichter P. (2013). Mutations in regulators of the epigenome and     their connections to global chromatin patterns in cancer. Nature     Reviews Genetics 14: 765-780. -   Taverna S D, Li H, Ruthenburg A J, Allis C D, Patel D J. (2007) How     chromatin-binding modules interpret histone modifications: lessons     from professional pocket pickers Nature Structural & Molecular     Biology 14, 1025-1040. -   Prinjha R K, Witherington J, Lee K. (2012) Place your BETs: the     therapeutic potential of bromodomains Trends in Pharmacological     Sciences 33, 3, 146-153. -   Biggar K L, Li S S-C. (2015) Non-histone protein methylation as a     regulator of cellular signalling and function. Nature Rev Mol Cell     Biol, 16, 5-17. -   Tough D F, Lewis H D, Rioja I, Lindon M J, Prinjha R K (2014).     Epigenetic pathways targets for the treatment of disease:     accelerating progress in the development of pharmacological tools:     IUPHAR Review 11. Br J Pharmacol 171: 4981-5010 -   Ciceri P, Müller S, O'Mahony A, Fedorov O, Filippakopoulos P, Hunt J     P, Lasater E A, Pallares G, Picaud S, Wells C, Martin S, Wodicka L     M, Shah N P, Treiber D K, Knapp S (2014) Dual kinase-bromodomain     inhibitors for rationally designed polypharmacology. Nature Chemical     Biology, 10, 305-312. -   Fu L, Tian M, Li X, Li J-J, Huang J L, Zhang Y, Liu B. (2015).     Inhibition of BET bromodomains as a therapeutic strategy for cancer     drug discovery. Oncotarget. 2015 Mar. 20; 6(8): 5501-5516. -   Ciro M, Prosperini E, Quarto M, Grazini U, Walfridsson J, McBlane F,     Nucifero P, Pacchiana G, Capra M, Christensen J, Helin K. (2009)     ATAD2 is a novel cofactor for MYC, overexpressed and amplified in     aggressive tumors. Cancer Res., 69: 8491-8498. -   Krakstad C, Tangen I L, Hoivik E A, Halle M K, Berg A, Wemer H M,     Salvesen, H B. (2015) ATAD2 overexpression links to enrichment of     B-MYB-translational signatures and development of aggressive     endometrial carcinoma. Oncotarget, 6: 28440-28452. -   Weidner-Glunde M, Ottinger M, Schulz T F. (2010) WHAT do viruses BET     on? Front Biosci. 15: 537-549. -   Blus B J, Wiggins K, S. (2011) Epigenetic virtues of chromodomains.     Critical Reviews in Biochemistry and Molecular Biology. 46, 6,     507-526 -   Cyr A R, Dormann F E. The redox basis of epigenetic modifications:     from mechanism to functional consequences. (2011) Antioxidant &     Redox Signaling, 15(2), 551-589. -   Copeland R A, Solomon M E, Richon V M. (2009) Protein methyl     transferase as a target class for drug discovery. Nat. Rev. Drug     Discovery, 8, 724-732. -   Shankar R S, Bahirvani A G, Rao V K, Bharathy N, Ow J R,     Taneja R. (2013) G9a a multipotent regulator of gene expression.     Epigenetics, 8:1, 16-22. -   Casciello F, Winloch K, Gannon F, Lee J S. (2015) Functional role of     G9a histone methyltransferase in cancer. Front. Immunol., 6:487-498. -   Fuhrmann J, Clancy K W, Thompson P R. (2015) Chemical biology of     protein arginine modifications in epigenetic regulation. Chem Rev.,     115, 5413-5461. -   Hojfelt J W, Agger K, Helin K. (2013) Histone lysine demethylases as     targets for anticancer therapy. Nat. Rev. Drug Discov. 917. -   Xiang Y, Zhu Z, Han G, Ye X, Xu B, Peng Z et al. (2007). JARID1B is     a histone H3 lysine 4 demethylase up-regulated in prostate cancer.     Proc Natl Acad Sci USA 104: 19226-19231. -   Couvelard A, Deschamps L, Rebours V, Sauvanet A, Gatter K, Pezzella     F et al. (2008). Overexpression of the oxygen sensors PHD-1, PHD-2,     PHD-3, and FIH is associated with tumor aggressiveness in pancreatic     endocrine tumors. Clin Cancer Res 14: 6634-6639. -   Roesch A, Fukunaga-Kalabis M, Schmidt E C, Zabierowski S E, Brafford     P A, Vultur A et al. (2010). A temporarily distinct subpopulation of     slow-cycling melanoma cells is required for continuous tumor growth.     Cell 141: 583-594. -   He J, Nguyen A T, Zhang Y (2011). KDM2b/JHDMlb, an H3K36me2-specific     demethylase, is required for initiation and maintenance of acute     myeloid leukemia. Blood 111: 3869-3880. -   Berry W L, Janknecht R (2013). KDM4/JMJD2 histone demethylases:     epigenetic regulators in cancer cells. Cancer Res 73: 2936-2942. -   Kogure M, Takawa M, Cho H-S, Toyokawa G, Hayashi K, Tsunoda T et al.     (2013). Deregulation of the histone demethylase JMJD2A is involved     in human carcinogenesis through regulation of the G1/S transition.     Cancer Lett 336: 76-84. -   Tzatsos A, Paskaleva P, Ferrari F, Deshpande V, Stoykova S, Contino     G et al. (2013). KDM2B promotes pancreatic cancer via polycomb     dependent and -independent transcriptional programs. J Clin Invest     123: 727-739. -   Adcock I M, Lee K Y (2006). Abnormal histone acetylase and     deacetylase expression and function in lung inflammation. Inflamm     Res 55:311-321. -   Avvakumov N, Cote J (2007). The MYST family of histone     acetyltransferases and their intimate links to cancer. Oncogene 26:     5395-5407. -   Grabiec A, Tak P, Reedquist K (2008). Targeting histone deacetylase     activity in rheumatoid arthritis and asthma as prototypes of     inflammatory disease: should we keep our HATs on? Arthritis Res Ther     10: 226. -   Ghizzoni M, Haisma H J, Maarsingh H, Dekker F J (2011). Histone     acetyltransferases are crucial regulators in NF-kB mediated     inflammation. Drug Discov Today 16: 504-511. -   Iyer A, Fairlie D P, Brown L (2011). Lysine acetylation in obesity,     diabetes and metabolic disease. Immunol Cell Biol 90: 39-46. -   Pirooznia S K and Elefant F (2013). Targeting specific HATs for     neurodegenerative disease treatment: translating basic biology to     therapeutic possibilities. Front Cell Neurosci 7: 30. -   Lee, S., et al., (2001) Combined angiotensin converting enzyme     inhibition and angiotensin AT(1) receptor blockade up-regulates     myocardial AT(2) receptors in remodeled myocardium post-infarction.     Cardiovasc Res, 51(1): 131-9. -   Rubin, B., Laffan R J, Kotler D G, O'Keefe E H, Demaio D A, Goldberg     M E (1978) S Q 14,225 (D-3-mercapto-2-methylpropanoyl-1-proline), a     novel orally active inhibitor of angiotensin I-converting enzyme. J     Pharmacol Exp Ther, 204(2): 271-80. -   Bunning, P., B. Holmquist, and J. F. Riordan, Substrate specificity     and kinetic characteristics of angiotensin converting enzyme.     Biochemistry, 1983. 22(1): p. 103-10. -   Hesselgesser, J., et al., Identification and characterization of     small molecule functional antagonists ofthe CCR1 chemokine receptor.     J Biol Chem, 1998. 273(25): p. 15687-92. -   Gong, X., Gong W, Kuhns D B, Ben-Baruch A, Howard O M, Wang J     M (1997) Monocyte chemotactic protein-2 (MCP-2) uses CCR1 and CCR2B     as its functional receptors. J Biol Chem, 272(18): 11682-5. -   Combadiere, C., Salzwedel K, Smith E D, Tiffany H L, Berger E A,     Murphy P M (1998) Identification of CX3CR1. A chemotactic receptor     for the human CX3C chemokine fractalkine and a fusion coreceptor for     HIV-1. J Biol Chem, 273(37): 23799-804. -   Grob, P. M., David E, Warren T C, DeLeon R P, Farina P R, Homon     C A. (1990) Characterization of a receptor for human     monocyte-derived neutrophil chemotactic factor/interleukin-8. J Biol     Chem, 265(14): 8311-6. -   Ahuja, S. K. and P. M. Murphy (1996) The CXC chemokines     growth-regulated oncogene (GRO) alpha, GRObeta, GROgamma,     neutrophil-activating peptide-2, and epithelial cell-derived     neutrophil-activating peptide-78 are potent agonists for the type B,     but not the type A, human interleukin-8 receptor. J Biol Chem,     271(34): 20545-50. -   Valenzuela-Femandez, A., Planchenault T, Baleux F, Staropoli I,     Le-Barillec K, Leduc D, Delaunay T, Lazarini F, Virelizier J L,     Chignard M, Pidard D, Arenzana-Seisdedos F. (2002) Leukocyte     elastase negatively regulates Stromal cell-derived factor-1     (SDF-1)/CXCR4 binding and functions by amino-terminal processing of     SDF-1 and CXCR4. J Biol Chem, 277(18): 15677-89. -   Dittadi, R., et al., Radioligand binding assay of epidermal growth     factor receptor: causes of variability and standardization of the     assay. Clin Chem, 1990. 36(6): p. 849-54. -   Muller-Enoch, D., E. Seidl, andH. Thomas, [6,7-Dihydroxycoumarin     (Aesculetin) as a substrate for catechol-o-methyltransferase     (author's transl)]. Z Naturforsch C, 1976. 31(5-6): 280-4. -   Tietge, U. J., Pratico D, Ding T, Funk C D, Hildebrand R B, Van     Berkel T, Van Eck M. (2005). Macrophage-specific expression of group     IIA sPLA2 results in accelerated atherogenesis by increasing     oxidative stress. J Lipid Res, 46(8): 1604-14. -   Montalibet, J., K. I. Skorey, and B. P. Kennedy (2005) Protein     tyrosine phosphatase: enzymatic assays. Methods 35(1): 2-8. -   Sun, Z. Y. and Z. H. Tu, A novel in vitro model to screen steroid 5     alpha-reductase inhibitors against benign prostatic hyperplasia.     Methods Find Exp Clin Pharmacol, 1998. 20(4): p. 283-7. -   Becker, K., Gromer, S., Schirmer, R. H., Müller, S. (2000)     Thioredoxin reductase as a pathophysiological factor and drug     target. Eur J Biochem, 267(20): 6118-25. -   Hatano, T., et al. (1990). Effects of interaction of tannins with     co-existing substances. VII. Inhibitory effects of tannins and     related polyphenols on xanthine oxidase. Chem Pharm Bull (Tokyo),     38(5): 1224-9. -   De Backer et al, (1993). Genomic cloning, heterologous expression     and pharmacological characterization of a human histamine H1     receptor. Biochem Biophys Res Commun 197(3): 1601-1608. -   Ruat et al., (1990). Reversible and irreversible labeling and     autoradiographic localization of the cerebral histamine H2 receptor     using [125I]iodinated probes. Proc Natl Acad Sci USA. 87(5):     1658-1662. -   Krueger et al, (2005). G protein-dependent pharmacology of histamine     H3 receptor ligands: evidence for heterogeneous active state     receptor conformations. J Pharmacol Exp Ther. 314(1): 271-281. -   Liu et al, (2001). Comparison of human, mouse, rat, and guinea pig     histamine H4 receptors reveals substantial pharmacological species     variation. J Pharmacol Exp Ther. 299(1): 121-130. -   Kubo and Strott, (1987). Differential activity of     3-hydroxy-3-methylglutaryl coenzyme A reductase in zones of the     adrenal cortex. Endocrinology. 120:214-221. -   Pufahl et al., (2007). Development of a fluorescence-based enzyme     assay of human 5-lipoxygenase. Anal Biochem. 364(2): 204-212. -   Mansuy et al., (1986). A new potent inhibitor of lipid peroxidation     in vitro and in vivo, the hepatoprotective drug anisylditholthione.     Biochem Biophys Res Comm. 135:1015-1021. -   Romano et al., (1993). Lipoxin synthase activity of human platelet     12-lipoxygenase. Biochem J. 296: 127-133. -   Urban et al., (1991). Comparative membrane locations and activities     of human monoamine oxidases expressed in yeast. FEBSLett. 286(1-2):     142-146. -   Svensson et al., (1987). Peroxidase and peroxidase-oxidase     activities of isolated human myeloperoxidase. Biochem J.     242:673-680. -   Yamamoto et al., (2006). A nonradioisotope, enzymatic assay for     2-deoxyglucose uptake in L6 skeletal muscle cells cultured in a     96-well microplate. Anal Biochem. 351(1): 139-45. -   Cobb, R. R., et al., (1992) Functional expression of soluble ICAM-1     by baculovirus-infected Sf9 cells. Biochem Biophys Res Commun,     185(3): 1022-33. -   Stoltenborg, J. K., Tsao P W, George H J, Bouchard P J, Wexler E J,     Hausner E A (1994). A fluorescent cellular adhesion assay using     insect cell produced human VCAM1. J Immunol Methods, 175(1): 59-68. -   Lenardo, M. J. and D. Baltimore (1989) NF-kappa B: a pleiotropic     mediator of inducible and tissue-specific gene control. Cell, 58(2):     227-9. -   Sen R., Baltimore D. (1986). Multiple nuclear factors interact with     immunoglobulin enhancer sequences. Cell, 46, 705-716. -   Pantano C., Reynaert N. L., van der Vliet A. V.,     Janssen-Heininger Y. M. W. (2006). Redox-sensitive kinases of the     nuclear factor κB signalling pathway. Antioxidants & Redox Signaling     8(9-10), 1791-1807. -   Brigelius-Flohê R., Flohê L. (2011). Basic principle and emerging     concepts in redox control of transcription factors. Antioxidants &     Redox Signaling, 15(8), 2335-2380. -   Ghosh G., Wang V. Y-F., Huang D-B., Fusco A. (2015). NF-κB     regulation: lessons from structures. Immunol. Rev. 246(1), 36-58. -   Akdis M., Aab A., Altunbulakli C., Azkur K., Costa R. A., Crameri     R., Duan S., Eiwegger T., Eljaszewicz A., Ferstl R., Frei R.,     Garbani M., Globinska A., Hess L., Huitema C., Kubo T., Komlosi Z.,     Konieczna P., Kovacs N., Kucuksezer U. C., Meyer N., Morita H.,     Olzhausen J., O'Mahony L., Perzer M., Prati M., Rabane A., Rhyner     C., Rinaldi A., Sokolowska M., Stanic B., Sugita K., Treis A., van     der Veen W., Wanke K., Wawrzyniak M., Wawrzyniak P., Wirz O. F.,     Zakzuk J. S, Akdis C. A. (2016). Interleukins (from IL-1 to IL-38),     interferons, transforming growth factor β, and TNF-α: receptors,     functions, and role in diseases. Fundamentals of allergy and     Immunology, 138, 4, 984-1010. -   Perkins N. D. (2012). The diverse and complex roles of NF-κB     subunits in cancer. Nature Reviews Cancer, 12, 121-133. -   Sethi G., Shanmugam M. K., Ramachandran L., Kumar A. P.,     Tergaonkar V. (2012). Multifacet link between cancer and     inflammation. Biosci. Rep. 32, 1-15. -   Parri M., Chiarugi P. (2013). Redox molecular machines involved in     tumour progression. Antioxidants & Redox Signaling, 19(15),     1828-1846. -   D'Ignazio L., Bandarra D., Rocha S. (2016). NF-κB cross talk in     immune responses. FEBS Journal 283, 413-424. -   Ludwig L. M., Nassin M. L., Hadji A., Labelle, J. L. (2016). Killing     two cells with one stone: Pharmacologic BCL-2 family targeting for     cancer cell death and immune modulation. Frontiers in Peadiatrics,     Vol 4, Article 135, page 1-13. -   Xia, M, Huang R, Witt K L, Southall N, Fostel J, Cho M H, Jadhav A,     Smith C S, Inglese J, Portier C J, Tice R R, Austin C P (2008)     Compound cytotoxicity profiling using quantitative high-throughput     screening. Environ Health Perspect. 116(3):284-91. -   Fallahi-Sichani, M., S. Honardejad, L. M. Heiser, J. W. Gray,     and P. K. Sorger (2013). Metrics other than potency reveal     systematic variation in responses to cancer drugs. Nat. Chem. Biol.     9: 708-714. -   Barretina, J., G. Caponigro, N. Stransky, K. Venkatesan, A. A.     Margolin, et al. (2012). The Cancer Cell Line Encyclopedia enables     predictive modelling of anticancer drug sensitivity. Nature 483:     603-607. -   Dierks E A, Stams K R, Lim H K, Cornelius G, Zhang H, Ball S E     (2001). A method for the simultaneous evaluation of the activities     of seven major human drug-metabolizing cytochrome P450s using an in     vitro cocktail of probe substrates and fast gradient liquid     chromatography tandem mass spectrometry. Drug Metab. Dispos., 29:     23-29. -   Mohammad R. H., Dugan, M. C., Mohamed, A. N., Almatchy, V. P.,     Flake, T. M., Dergham, S. T., Shields, A. F., Al-Katib, A. A.,     Vaitkevicius, V. K. and Sarkar, F. H (1998). Establishment of a     human pancreatic tumor xenograft model: Potential application for     preclinical evaluation of novel therapeutic agents. Pancreas     16:19-25. -   “Guide for the Care and Use of Laboratory Animals: Eighth Edition”     (2011), The National Academies Press, Washington, D.C. 

What is claimed is:
 1. A method of treating a disease or disorder selected from the group consisting of a cancer, a skin disease, or a skin disorder, the method comprising: administering a therapeutically effective amount of a compound according to a compound of Formula (I),

a geometric isomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein: R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O; R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(P)C₄₋₅alkadienyl or ═O; R₃ is independently H, OH, OC₁₋₅alkyl or OC(O)C₁₋₅alkyl or ═O, no more than one of R₁, R₂ and R₃ can be H; A-B is CH═C or CH₂—CH; and (a) X is CH₂, or C(O), and R₄ is OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; or (b) X and R₃ are directly joined with each other to form a six membered ring, provided that X is CH, CH₂, or C(O), and R₃ is O, wherein when X is CH then R₄ is OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl, or when X is CH₂ or C(O) then R₄ is absent.
 2. The method according to claim 1, wherein, (i) when R₂ is H, X is not CH₂; and (ii) when R₂ is H, and when X and R₃ form a six membered ring, then A-B is CH═C.
 3. The method according to claim 1, wherein the compound is (i) a compound of Formula (Ia):

or (ii) a compound of Formula (Ic):

or (iii) a compound of Formula (Ii):


4. The method according to claim 1, wherein A-B is CH═C.
 5. The method according to claim 1, wherein the compound is selected from the group consisting of:


6. The method according to claim 5, wherein the compound is:


7. The method according to claim 5, wherein the compound is:


8. The method according to claim 1, wherein the compound is isolated from propolis, and wherein the propolis originates from plants of the Myoporum genus.
 9. The method according to claim 8, wherein the propolis originates from Myoporum insulare.
 10. The method as defined in claim 1, wherein the compound is isolated from the resin, gum or exudate of Myoporum genus.
 11. The method according to claim 10, wherein the compound is isolated from the resin, the gum, or the exudate of Myoporum insulare.
 12. The method according to claim 1, wherein the skin disease or the skin disorder is selected from the group consisting of eczema, psoriasis, acne, a wound, a scar, inflammation, a burn, sunburn, skin damage and skin irritation.
 13. The method according to claim 1, wherein the disease or the disorder is the cancer, and wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.
 14. The method according to claim 13, wherein the cancer is of the central nervous system and is a glioma.
 15. The method according to claim 13, wherein the cancer is of the central nervous system and is a medulloblastoma.
 16. The method according to claim 13, wherein the cancer is of the central nervous system and is a neuroblastoma.
 17. The method according to claim 13, wherein the cancer is the lung cancer and the lung cancer is a non-small cell lung cancer.
 18. The method according to claim 13, wherein the cancer is the lung cancer and the lung cancer is a small cell lung cancer.
 19. The method according to claim 13, wherein the cancer is the carcinoma and the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.
 20. The method according to claim 13, wherein the cancer is the sarcoma and the sarcoma is a liposarcoma, a rhabdomyosarcoma, or a fibrosarcoma.
 21. The method according to claim 13, wherein the cancer is the sarcoma and the sarcoma is a soft tissue sarcoma.
 22. The method according to claim 21, wherein the soft tissue sarcoma is a soft tissue osteosarcoma.
 23. The method according to claim 13, wherein the cancer is the lymphoma and the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.
 24. A method of manufacturing a medicament for treating a disease or disorder selected from the group consisting of a cancer, a skin disease, or a skin disorder, comprising, dissolving an effective amount of a compound according to Formula (I) for treating the cancer, the skin disease or the disorder with a pharmaceutically acceptable excipient, wherein Formula (I) is

a geometric isomer, stereoisomer, or a pharmaceutically acceptable salt thereof, wherein: R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O; R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl, OC(O)C₄₋₅alkadienyl or ═O; R₃ is independently H, OH, OC₁₋₅alkyl or OC(O)C₁₋₅alkyl or ═O; and no more than one of R₁, R₂ and R₃ can he H; A-B is CH═C or CH₂—CH; and (a) X is CH₂, or C(O), and R₄ is OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl; or (b) X and R₃ are directly joined with each other to form a six-membered ring, and X is CH, CH2, or C(O), and R3 is O, wherein when X is CH then R₄ is OH, OC₁₋₅alkyl, OCH₂Ph, OC₂₋₅alkenyl, OC₄₋₅alkadienyl, OC(O)C₁₋₅alkyl, OC(O)Ph, OC(O)C₂₋₅alkenyl or OC(O)C₄₋₅alkadienyl, or when X is CH2 or C(O) then R4 is absent.
 25. The method according to claim 24, wherein (i) when R₂ is H then, X is not CH₂; and (ii) when R₂ is H, and X and R₃ form a six membered ring, then A-B is CH═C.
 26. The method according to claim 24, wherein: (i) the compound is a compound of Formula (Ia):

or (ii) a compound of Formula (Ic):

or (iii) a compound of Formula (Ii):


27. The use method according to claim 24, wherein A-B is CH═C.
 28. The method according to claim 24, wherein the compound is a compound selected from the group consisting of:


29. The method according to claim 24, wherein the skin disease or the skin disorder is the cancer and the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.
 30. The method according to claim 24, wherein the disease or the disorder is the skin disease or the skin disorder and the skin disease or the skin disorder is selected from the group consisting of: eczema, psoriasis, acne, a wound, a scar, inflammation, a burn, sunburn, skin damage and skin irritation.
 31. The method according to claim 1, wherein: R₁ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph, or ═O; R₂ is independently H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O; R₃ is independently H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O; no more than one of R₁, R₂ and R₃ can be H; A-B is CH═C or CH2-CH; and (a) X is CH₂, and R₄ is OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, or OC(O)Ph, or (b) X and R₃ are directly joined with each other to form a six membered ring, provided that X is CH or CH2 and R3 is O, wherein when X is CH then R4 is OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, or OC(O)Ph, or when X is CH2 then R4 is absent.
 32. The method of claim 1, wherein: X is CH₂; R₁ is independently OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O; R₂ is independently OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O; R₃ is H; R₄ is OH, OC₁₋₅alkyl, or OC(O)C₁₋₅alkyl; and A-B is CH═C or CH₂—CH.
 33. The method of claim 1, wherein: X is CH₂; R₁ is independently OH or OC₁₋₅alkyl; R₂ is independently OH or OC₁₋₅alkyl; R₃ is H; R₄ is OH or OC₁₋₅alkyl; and A-B is CH═C.
 34. The method of claim 24, wherein: R₁ is independently selected from H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph, or ═O; R₂ is independently selected from H, OH, OC₁₋₅alkyl, OCH₂Ph, C(O)C₁₋₅alkyl, OC(O)Ph, or ═O; R₃ is independently selected from H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O; wherein, no more than one of R₁, R₂ and R₃ can be H; A-B is CH═C or CH₂—CH; and (a) X is CH₂ and R4 is OH, OC₁₋₅alky, OCH₂Ph, OC(O)C₁₋₅alkyl, or OC(O)Ph, or (b) X and R3 are directly joined with each other to form a six membered ring, provided that X is CH or CH2 and R3 is O, wherein when X is CH then R4 is OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, or OC(O)Ph, or when X is CH2 then R4 is absent.
 35. The method according to claim 24, wherein: R₁ is independently selected from H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O; R₂ is independently selected from H, OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl, OC(O)Ph or ═O, R₃ is independently selected from H, OH, OC₁₋₅alkyl, OC(O)C₁₋₅alkyl or ═O; wherein, no more than one of R₁, R₂ and R₃ can be H; A-B is CH═C or CH₂—CH; and (a) X is CH₂ and R₄ is OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph; or (b) X and R₃ are directly joined with each other to form a six-membered ring, provided that X is CH or CH2 and R3 is O, wherein when X is CH then R₄ is OH, OC₁₋₅alkyl, OCH₂Ph, OC(O)C₁₋₅alkyl or OC(O)Ph, or when X is CH2 then R4 is absent.
 36. The method of claim 24, wherein: X is CH₂; R₁ is OH or OC₁₋₅alkyl; R₂ is OH or OC₁₋₅alkyl; R₃ is H; R₄ is OH or OC₁₋₅alkyl; and A-B is CH═C. 